Cannabis extracts and uses thereof

ABSTRACT

The present disclosure concerns a group of cannabinoid compounds defined by formulas (I) to (IV), wherein R 1  is —H or —COOH, for the first time isolated and fully characterized in structure, absolute stereochemistry by the present applicant. Methods of isolation, characterization, stereoselective synthesis, biological activity, pharmaceutical compositions and therapeutic applications of the present compounds as modulators of the cannabinoid CB1 receptor are also object of the disclosure.

TECHNICAL FIELD

The present disclosure relates generally to Cannabis extracts, inparticular cannabinoid compounds derived from these extracts and relateduses thereof.

BACKGROUND

What is needed are Cannabis extracts and compounds derived from Cannabisand related uses thereof.

SUMMARY

The present disclosure concerns a group of cannabinoid compounds for thefirst time isolated and fully characterized in structure, absolutestereochemistry by the present applicant; the biological activity ofthese compounds and their possible therapeutic applications are alsoexperimentally investigated and disclosed herein.

The compounds object of the present disclosure are selected from thegroup consisting of:

-   -   (−)trans (1R,6R) cannabidibutol [CBDB] of formula (I),    -   (−)trans (1R,6R) Δ⁹ tetrahydrocannabutol [Δ⁹ THCB] of formula        (II),    -   (−)trans (1R,6R) Δ⁹ tetrahydrocannabiphorol [Δ⁹ THCP] of formula        (III),    -   (−)trans (1R,6R) cannabidiphorol [CBDP] of formula (IV)

wherein R₁ is —H.

The disclosure also includes the corresponding acid derivatives:

-   -   (−)trans (1R,6R) cannabidibutolic acid [CBDBA] of formula (I),    -   (−)trans (1R,6R) Δ⁹ tetrahydrocannabutolic acid [Δ⁹ THCBA] of        formula (II),    -   (−)trans (1R,6R) Δ⁹ tetrahydrocannabiphorolic acid [Δ⁹ THCPA] of        formula (III),    -   (−)trans (1R,6R) cannabidiphorolic acid [CBDPA] of formula (IV),    -   wherein R₁ is —COOH.

Prior to the present disclosure, the full structure of some of thecompounds of formulas (I) to (IV) were not disclosed, while others werenever described nor characterized at all; moreover, the biologicalactivity and therapeutic potential for each of the present compounds,expressed as binding affinity to the cannabinoid CB1 receptor, was neverinvestigated prior to this disclosure.

The compounds of formulas (I) to (IV) occur in small amounts in natureand can be isolated form hemp (Cannabis sativa L.), in particular fromhemp cannabinoid active agents (CBD) and Δ⁹ tetrahydrocannabinol (Δ⁹THC, or simply THC), wherein they may generally occur is small amountsas impurities. Non-limitative procedures for the isolation of thecompounds of formulas (I) to (IV) from their natural sources areillustrated in the experimental part of this application.

The present disclosure is also directed to methods for thestereoselective synthesis of the compounds of formulas (i) to (IV). Theso-obtained products have been used herein to confirm, by comparison,the full stereochemistry of their corresponding natural counterparts;however, the disclosure is not limited to this use and the syntheticstereoselective procedures disclosed herein can be used in general tomanufacture the present compounds of formulas (I) to (IV) for anypurposes, in particular for industrial production, in alternative totheir extraction from hemp or hemp cannabinoids. The present syntheticmethods also make available, for the first time in enabling way, thecorresponding compounds with their complete stereochemicalconfiguration.

The method to produce the cannabinoid compound CBDB of formula (I)comprises reacting (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enolwith 5-butylbenzene-1,3-diol according to scheme (I) in presence of anacidic catalyst, obtaining CBDB.

The above reaction (Friedel-Craft allylation) can be applied likewise tothe production of the cannabinoid compound CBDP of formula (IV), withthe difference that the compound to be reacted with(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol is5-heptylbenzene-1,3-diol according to the following scheme (II); thereaction is also performed in presence of an acidic catalyst, obtainingin this case CBDP.

In both schemes (I) and (II) the acidic catalyst is preferablyp-toluensulphonic acid. Further, in both schemes (I) and (II), thereaction is preferably performed under inert atmosphere in a halogenatedorganic solvent at a temperature of −10±5° C., for a time ranging from30 to 90 min. The halogenated solvent is more preferablydichloromethane.

The method to produce the cannabinoid compound Δ⁹ THCB of formula (II)comprises reacting the CBDB of the above described formula (I) withhydrochloric acid obtaining the intermediate compound (−)trans HCl-THCBof formula (V).

The intermediate compound of formula (V) is then treated with a basiccompound, obtaining Δ⁹ THCB.

In a preferred embodiment, the reagent CBDB is not used in pure form,but in a mixture with its thermodynamically more stable conversionproduct (−)trans Δ⁸ THCB of formula (VII)

The mixture of CBDB with compound (VII) is easily obtained in a processof stereoselective synthesis of CBDB as previously described, whereinCBDB is not isolated immediately, but is allowed to remain in thereaction mixture, as long as a convenient amount of compound (VII), e.g.40% or more of the original CBDB, is converted into compound (VII).

The above described method to produce the cannabinoid compound Δ⁹ THCBof formula (II) can be applied likewise to the production of thecannabinoid compound Δ⁹ THCP of formula (II), with the difference thatthe compound to be reacted with hydrochloric acid is the CBDP of formula(IV) previously described, obtaining in this case the intermediatecompound (−)trans HCl-THCP of formula (VI),

The intermediate compound (VI) is then treated with a basic compound,obtaining Δ⁹ THCP.

Also in this case, in a preferred embodiment, the reagent CBDP is notused in pure form, but in a mixture with its thermodynamically morestable conversion product (−)trans Δ⁸ THCP of formula (VIII)

The mixture of CBDP with compound (VIII) is easily obtained in a processof stereoselective synthesis of CBDP as previously described, whereinCBDP is not isolated immediately, but is allowed to remain in thereaction mixture, as long as a convenient amount of compound (VIII),e.g. 40% or more of the original CBDP, is converted into compound(VIII).

In an even preferred embodiment, the mixture reacted with hydrochloricacid is exclusively or almost exclusively made of compound (VIII).

In both synthesis of Δ⁹ THCB e Δ⁹ THCP, the reaction with hydrochloricacid is preferably performed under catalysis of ZnCl₂. Furthermore, inboth synthesis of Δ⁹ THCB e Δ⁹ THCP, the basic compound reacted with therespective intermediates (V) and (VI) is preferably potassium amylate.

The above referred methods of stereoselective synthesis have beendescribed for the compounds of formulas (I) to (IV) in which the groupR₁ is —H (hydrogen). The synthesis of the corresponding acid derivativesin which the group R₁ is —COOH (carboxy) can be realized as describedabove, using the corresponding equivalent reagents in which R₁ is —COOH,in particular using 6-carboxy 5-butylbenzene-1,3-diol in the aboveScheme (I) or 6-carboxy 5-heptylbenzene-1,3-diol in the above Scheme(II).

Non-limitative examples of synthesis of the compounds of formula (I) to(IV), developed according to the above rules, are presented in fullextension in the experimental section.

The present compounds are characterized by affinity to a cannabinoidreceptor, in particular the cannabinoid receptor CB1. In some instances,the compounds are exclusively affine to the CB1 receptor; in otherinstances, they are mainly affine to the CB1, with a minor affinity tothe CB2 receptor. In all instances, the CB1 affinity is dominant, withthe consequence that the compounds can be used in the modulation of theCB1 receptor. The modulation can be of agonist or antagonist type;preferably it is of the agonist type.

In particular, it has been found that the length of the alkyl chain ofthe resorcinyl moiety present in formulas (I) to (IV) positivelycorrelates with the potency/specificity of CB1 binding affinity, asconfirmed by the fact that heptyl derivatives like the compound (III)showed the highest binding affinity to CB1. This finding was furthersupported by the evidence of docking pose studies in the CB1 receptor,also reported in the experimental section, whereby the full occupancy ofthe binding pocket of the hCB1 receptor was obtained only in case of theheptyl derivatives of formulas (III) and (IV).

Based on these findings, a further object of the present disclosure is amethod of treating or preventing a disease mediated by the CB1 receptorcomprising administering a compound claim 1 to a patient in needthereof.

A further object of the disclosure is a pharmaceutical compositioncomprising a compound of claim 1 in presence of one or morepharmaceutically acceptable excipients.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing summary and the following detailed description are betterunderstood when read in conjunction with the appended drawings.Exemplary embodiments are shown in the drawings; however, it isunderstood that the embodiments are not limited to the specificstructures depicted herein.

FIG. 1 shows structure and names (IUPAC, INN and abbreviation) ofcannabidiol (CBD), cannabidibutol (CBDB), cannabidivarin (CBDV), mainby-products of synthesis of CBD (1 and 2) and ibuprofen (used asinternal standard for HPLC-UV method), according to an exemplaryembodiment of the present disclosure.

FIG. 2 shows total Ion Chromatogram (TIC) of a CBD authentic sample,according to an exemplary embodiment of the present disclosure. The mailpeak is identified as CBD, the other two peaks at 2.54 and 3.24 min areimpurity 1 and impurity 2 respectively.

FIG. 3 shows extracted Ion Chromatograms (EIC) of CBD, CBDB and CBDV inpositive (top) and negative (bottom) ionization mode, according to anexemplary embodiment of the present disclosure. Area of the peaks wereobtained by extracting the exact mass (Δppm=2) of CBD ([M+H]⁺ 315.2314,[M−H]⁻ 313.2179), CBDB ([M+H]⁺ 301.2157, [M−H]⁻ 299.2016) and CBDV([M+H]⁺ 287.2002, [M−H]⁻ 285.1861). The chromatograms show the exactmatch of CBD and CBDV in samples with authentic analytical standards andthe match of synthetic CBDB with natural isolated CBDB.

FIG. 4(A) shows match of MS/MS spectra of CBD in authentic samples andreference analytical standard in positive ionization mode byUHPLC-HESI-Orbitrap, according to an exemplary embodiment of the presentdisclosure.

FIG. 4(B) shows match of MS/MS spectra of CBD in authentic samples andreference analytical standard in negative ionization mode byUHPLC-HESI-Orbitrap, according to an exemplary embodiment of the presentdisclosure.

FIG. 5(A) shows match of MS/MS spectra of CBDB in authentic samples andreference analytical standard (synthetic CBDB) in positive ionizationmode by UHPLC-HESI-Orbitrap, according to an exemplary embodiment of thepresent disclosure.

FIG. 5(B) shows match of MS/MS spectra of CBDB in authentic samples andreference analytical standard (synthetic CBDB) in negative ionizationmode by UHPLC-HESI-Orbitrap, according to an exemplary embodiment of thepresent disclosure.

FIG. 6(A) shows match of MS/MS spectra of CBDV in authentic samples andreference analytical standard in positive ionization mode byUHPLC-HESI-Orbitrap, according to an exemplary embodiment of the presentdisclosure, according to an exemplary embodiment of the presentdisclosure.

FIG. 6(B) shows match of MS/MS spectra of CBDV in authentic samples andreference analytical standard in negative mode by UHPLC-HESI-Orbitrap,according to an exemplary embodiment of the present disclosure,according to an exemplary embodiment of the present disclosure.

FIG. 7 shows fragmentation pattern of CBD, CBDB and CBDV in positiveionization mode, according to an exemplary embodiment of the presentdisclosure. The chemical protonated structure is indicated for the mainfragments. Red dashed lines indicate the matching fragments between thethree cannabinoids. The blue dashed box includes the unchanged fragmentsbelonging to the terpene moiety, according to an exemplary embodiment ofthe present disclosure.

FIG. 8 shows fragmentation pattern of CBD, CBDB and CBDV in negativeionization mode. The chemical ionized structure is indicated for themain fragments. Red dashed lines indicate the matching fragments betweenthe three cannabinoids, according to an exemplary embodiment of thepresent disclosure.

FIG. 9(A) shows ¹H-NMR spectra and peaks assignment for CBDB, accordingto an exemplary embodiment of the present disclosure.

FIG. 9(B) shows ¹³C-NMR spectra and peaks assignment for CBDB, accordingto an exemplary embodiment of the present disclosure.

FIG. 10(A) shows superimposition of ¹H-NMR of isolated natural(ext-CBDB, red spectra) and synthesized (syn-CBDB, blue spectra),according to an exemplary embodiment of the present disclosure.

FIG. 10(B) shows superimposition of ¹³C-NMR spectra of isolated natural(ext-CBDB, red spectra) and synthesized (syn-CBDB, blue spectra),according to an exemplary embodiment of the present disclosure.

FIG. 11 shows structure of the main cannabinoids THC and CBD, theiracidic precursors, THCA and CBDA, and their respective propyl and butylhomologues, according to an exemplary embodiment of the presentdisclosure.

FIGS. 12(A) and 12(B) collectively show UHPLC-HRMS chromatograms of anFM2 ethanolic extracts.

FIG. 12(A) shows the extracted ion chromatograms (EICs) of the acidicforms before (native FM2) and after decarboxylation (decarboxylatedFM2): EICs were chosen based on the exact mass calculated forC₂₂H₃₀O₄(THCA and CBDA), C₂₁H₂₈O₄(THCBA and CBDBA) and C₂₀H₂₆O₄ (THCVAand CBDVA).

FIG. 12(B) shows the EICs of the neutral species before (native FM2) andafter decarboxylation (decarboxylated FM2): EICs were chosen based onthe exact mass calculated for C₂₁H₃₀O₂(THC and CBD), C₂₀H₂₈O₂(THCB andCBDB) and C₁₉H₂₆O₂(THCV and CBDV), according to an exemplary embodimentof the present disclosure. Each peak is labelled with compound name andpeak area.

FIG. 13 shows docking pose of (−)-trans-Δ⁹-THCV (a, orange sticks),(−)-trans-Δ⁹-THCB (B, deep blue sticks) and (−)-trans-Δ⁹-THC (C, purplesticks) in complex with CB1 receptor (PDB ID: 5XRA, deep teal cartoon),according to an exemplary embodiment of the present disclosure.Important amino acidic residues are reported in deep teal sticks. H-bondare reported in yellow dotted line. Heteroatoms are colored in red(oxygen), yellow (sulphur).

FIG. 14(A) shows the effect of Δ⁹-THCB (2, 3 and 5 mg/kg, i.p.) in theformalin test in mice. The total time of the nociceptive response wasmeasured every 5 min and expressed in min (see experimental section),according to an exemplary embodiment of the present disclosure. Data arerepresented as means±S.E.M. (n=5). * and ** indicate statisticallysignificant differences vs. Veh/form, p<0.05 and p<0.01 respectively.

FIG. 14(B) shows the effect of AM251 (0.5 mg/Kg i.p.) Δ⁹-THCB (3 mg/kg,i.p.) in the formalin test in mice, according to an exemplary embodimentof the present disclosure. The total time of the nociceptive responsewas measured every 5 min and expressed in min (see methods). Data arerepresented as means±S.E.M. (n=5). * and ** indicate statisticallysignificant differences vs. Veh/form, p<0.05 and p<0.01 respectively. º,ºº and ººº indicate statistically significant differences vs THCB,p<0.05, p<0.01 and p<0.001, respectively, according to an exemplaryembodiment of the present disclosure.

FIG. 14(C) shows the effect of AM630 (1 mg/Kg i.p.) Δ⁹-THCB (3 mg/kg,i.p.) in the formalin test in mice, according to an exemplary embodimentof the present disclosure. The total time of the nociceptive responsewas measured every 5 min and expressed in min (see methods). Data arerepresented as means±S.E.M. (n=5). * and ** indicate statisticallysignificant differences vs. Veh/form, p<0.05 and p<0.01 respectively. º,ºº and ººº indicate statistically significant differences vs THCB,p<0.05, p<0.01 and p<0.001, respectively, according to an exemplaryembodiment of the present disclosure.

FIGS. 15(A), 15(B), 15(C), 15(D), 15(E) and 15(F), collectively, showthe effect of THCB (10 and 20 mg/kg, i.p.) in the tetrad test, accordingto an exemplary embodiment of the present disclosure.

FIG. 15(A) shows a timeline of the tetrad procedure from Δ⁹-THCBadministration, according to an exemplary embodiment of the presentdisclosure.

FIG. 15(B) shows representative examples of movement path in vehicle orΔ⁹-THCB, according to an exemplary embodiment of the present disclosure.

FIG. 15(C) shows motor activity (distance, cm) in the OFT, according toan exemplary embodiment of the present disclosure.

FIG. 15(D) shows body temperature (change in body temperature, ° C.),according to an exemplary embodiment of the present disclosure.

FIG. 15(E) shows catalepsy (latency for moving, s) in the bar test,according to an exemplary embodiment of the present disclosure.

FIG. 15(F) shows analgesia (latency to first sign of pain, s) in the hotplate test, according to an exemplary embodiment of the presentdisclosure. Data in FIGS. 15(A)-(E) are represented as mean±SEM of 4mice per group. * indicates significant differences compared to 0(vehicle injection); specifically: *p<0.05, **p<0.01, ***p<0.001 vs.vehicle and the Kruskall-Wallis test followed by Dunn's post hoc testswas used for statistical analysis.

FIG. 16 shows effect of THCB (PLC14) or CBDB (PLC4), at different doses,on the two phases of formalin test, according to an exemplary embodimentof the present disclosure.

FIGS. 17(A), 17(B), 17(C) and 17(D) collectively, show UHPLC-HRMSidentification of (−)-trans-CBDP and (−)-trans-Δ⁹-THCP, according to anexemplary embodiment of the present disclosure.

FIG. 17(A) shows extracted ion chromatograms (EIC) of CBDP and Δ⁹-THCPfrom a standard mixture at 25 and 10 ng/mL respectively FIG. 17(a) andfrom the native (red plot) and decarboxylated (black plot) FM2.

FIG. 17(B) shows extracted ion chromatograms (EIC) of CBDP and Δ⁹-THCPfrom a standard mixture at 25 and 10 ng/mL respectively FIG. 17(a) andfrom the decarboxylated (black plot) FM2.

FIG. 17(C) shows a comparison of the high-resolution fragmentationspectra of synthetic and natural CBDP and Δ⁹-THCP in both positive(ESI+) and negative (ESI−) mode, according to an exemplary embodiment ofthe present disclosure.

FIG. 17(D) shows a comparison of the high-resolution fragmentationspectra of synthetic and natural CBDP and Δ⁹-THCP in both positive(ESI+) and negative (ESI−) mode, according to an exemplary embodiment ofthe present disclosure.

FIGS. 18(A), 18(B), 18(C), 18(D), 18(E), 18(F), and 18(G), collectively,show synthesis and spectroscopic characterization of (−)-trans-CBDP and(−)-trans-Δ⁹-THCP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(A) shows reagents and conditions: a) 5-heptylbenzene-1,3-diol(1.1 eq.), pTSA (0.1 eq.), CH₂Cl₂, r.t., 90 min.; b)5-heptylbenzene-1,3-diol (1.1 eq.), pTSA (0.1 eq.), DCM, r.t., 48 h; c)pTSA (0.1 eq.), CH₂Cl₂, r.t., 48 h; d) ZnCl₂ (0.5 eq.), 4N HCl indioxane (1 mL per 100 mg of Δ⁸-THCP), dry CH₂Cl₂, argon, 0° C. to r.t.,2 h. e) 1.75M potassium t-amylate in toluene (2.5 eq.), dry toluene,argon, −15° C., 1 h, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(B) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-CBDP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(C) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-CBDP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(D) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-CBDP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(E) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-Δ⁹-THCP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(F) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-Δ⁹-THCP, according to an exemplary embodiment of the presentdisclosure.

FIG. 18(G) shows superimposition of ¹H, ¹³C NMR and circular dichroismspectra for natural (red line) and synthesized (blue line)(−)-trans-Δ⁹-THCP, according to an exemplary embodiment of the presentdisclosure.

FIGS. 19(A) and 19(B) show in vitro activity and docking calculation ofΔ⁹-THCP, according to an exemplary embodiment of the present disclosure.FIG. 19(A) shows binding affinity (K_(i)) of the four homologues ofΔ⁹-THC against human CB₁ and CB₂ receptors, according to an exemplaryembodiment of the present disclosure.

FIG. 19(B) shows dose-response studies of Δ⁹-THCP against hCB₁ (in blue)and hCB₂ (in grey). All experiments were performed in duplicate anderror bars denote s.e.m. of measurements, according to an exemplaryembodiment of the present disclosure.

FIG. 19(C) shows docking pose of (−)-rans-Δ⁹-THCP (blue sticks), incomplex with hCB₁ receptor (PDB ID: 5XRA, orange cartoon), according toan exemplary embodiment of the present disclosure. Key amino acidicresidues are reported in orange sticks. H-bond are reported in yellowdotted line. Heteroatoms are color-coded: oxygen in red, nitrogen inblue and sulphur in yellow.

FIG. 19(D) shows binding pocket of hCB₁ receptor, highlighting thepositioning of the heptyl chain within the long hydrophobic channel ofthe receptor (yellow dashed line), according to an exemplary embodimentof the present disclosure. The side hydrophobic pocket is bordered inmagenta.

FIGS. 20(A), 20(B), 20(C), 20(D), 20(E) and 20(F), collectively, showdose-dependent effects of Δ⁹-THCP administration (2.5, 5, or 10 mg/kg,i.p.) on the tetrad phenotypes in mice in comparison to vehicle,according to an exemplary embodiment of the present disclosure.

FIG. 20(A) shows time schedule of the tetrad tests in minutes fromΔ⁹-THCP or vehicle administration, according to an exemplary embodimentof the present disclosure.

FIG. 20(B) shows locomotion decrease induced by Δ⁹-THCP administrationin the open field test, according to an exemplary embodiment of thepresent disclosure.

FIG. 20(C) shows locomotion decrease induced by Δ⁹-THCP administrationin the open field test, according to an exemplary embodiment of thepresent disclosure.

FIG. 20(D) shows decrease of body temperature after Δ⁹-THCPadministration; the values are expressed as the difference between thebasal temperature (i.e., taken before Δ⁹-THCP or vehicle administration)and the temperature measured after Δ⁹-THCP or vehicle administration,according to an exemplary embodiment of the present disclosure.

FIG. 20(E) shows increase in the latency for moving from the catalepsybar after Δ⁹-THCP administration, according to an exemplary embodimentof the present disclosure.

FIG. 20(F) shows increase in the latency after the first sign of painshown by the mouse in the hot plate test following Δ⁹-THCPadministration, according to an exemplary embodiment of the presentdisclosure. Data in FIGS. 20(a)-(f) are represented as mean±SEM of 5mice per group, * indicate significant differences compared to 0(vehicle injection), respectively, *p<0.05, **p<0.01, ***p<0.001 versusT7 0 mg/kg (vehicle), and the Kruskall-Wallis test followed by Dunn'spost hoc tests.

DETAILED DESCRIPTION

The terminology used in the present disclosure is for the purpose ofdescribing particular exemplary embodiments only and is not intended tobe limiting. As used in the description of the embodiments of thedisclosure and the appended claims, the singular forms “a,” “an,” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

The term “and/or,” as used herein, refers to and encompasses any and allpossible combinations of one or more of the associated listed items.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of a component, time, temperature, and the like, ismeant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% ofthe specified amount. Unless otherwise defined, all terms, includingtechnical and scientific terms used in the description, have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs.

As used herein, the term “treating” or “treatment” refers to any indiciaof success in the treatment or amelioration of an injury, pathology,condition, or symptom (e.g., pain), including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter including, e.g., theresult of a physical examination.

As used herein, the term “administering” refers to oral, topical,parenteral, intravenous, intraperitoneal, intramuscular, intralesional,intranasal, subcutaneous, or intrathecal administration to a subject, aswell administration as a suppository or the implantation of aslow-release device, e.g., a mini-osmotic pump, in the subject.

As referred above, the present compounds are characterized by affinityto a cannabinoid receptor, in particular the cannabinoid receptor CB1.In some instances, the compounds are exclusively affine to the CB1receptor; in other instances, they are mainly affine to the CB1, with aminor affinity to the CB2 receptor. In all instances, the CB1 affinityis dominant, with the consequence that the compounds can be used in themodulation of the CB1 receptor. The modulation can be of agonist orantagonist type; preferably it is of the agonist type.

There is no strict limitation as to the type of diseases mediated by theCB1 receptor being target of the present treatment. A non-limitativelist thereof includes: metabolic syndromes such as type 2 diabetes,dyslipidemia, and obesity; eating disorders; constipation;cardiovascular diseases or disorders such as hypertension, congestiveheart failure, cardiac hypertrophy, peripheral artery disease,cerebrovascular accidents, atherosclerosis, stroke, myocardialinfarction, and cardiotoxicity associated with chemotherapy; fatty liverdisease (steatohepatitis) and non-alcoholic fatty liver disease; kidneydisease; diseases or disorders characterized by an addiction componentsuch as smoking addiction or withdrawal, alcohol addiction orwithdrawal, and drug addiction or withdrawal; bone diseases or disorderssuch as osteoporosis, Paget's disease of bone, and bone cancer; breastcancer; inflammatory diseases such as neuropathy and neuro-inflammatorydisorders; autoimmune diseases such as rheumatoid arthritis,inflammatory bowel disease, and psoriasis; psychiatric diseases ordisorders such as anxiety, mania, schizophrenia; disorders or diseasesassociated with memory impairment and/or loss of cognitive function suchas Parkinson's disease, Alzheimer's disease and dementia; migraine;multiple sclerosis and Guillain-Barre syndrome; epilepsy; asthma.

The present compounds can be administered at any suitable dose in themethods of the disclosure. In general, the compounds are administered ata dose ranging from about 0.1 milligrams to about 1000 milligrams perkilogram of a subject's body weight (i.e., about 0.1-1000 mg/kg). Thedose of a compound can be, for example, about 0.1-1000 mg/kg, or about1-500 mg/kg, or about 25-250 mg/kg, or about 50-100 mg/kg. The dose canbe about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg/kg.

The dosages can be varied depending upon the requirements of thepatient, the severity of the disorder being treated, and the particularformulation being administered. The dose administered to a patientshould be sufficient to result in a beneficial therapeutic response inthe patient. The size of the dose will also be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of the drug in a particular patient. Determination ofthe proper dosage for a particular situation is within the skill of thetypical practitioner. The total dosage can be divided and administeredin portions over a period of time suitable to treat to the condition ordisorder.

Administration can be conducted for a period of time which will varydepending upon the nature of the particular disorder, its severity andthe overall condition of the patient. Administration can be conducted,for example, hourly, every 2 hours, three hours, four hours, six hours,eight hours, or twice daily including every 12 hours, or any interveninginterval thereof. Administration can be conducted once daily, or onceevery 36 hours or 48 hours, or once every month or several months.Following treatment, a patient can be monitored for changes in his orher condition and for alleviation of the symptoms of the disorder. Thedosage can either be increased in the event the patient does not respondsignificantly to a particular dosage level, or the dose can be decreasedif an alleviation of the symptoms of the disorder is observed, or if thedisorder has been ablated, or if unacceptable side effects are seen witha particular dosage.

A therapeutically effective amount of a compound of the disclosure canbe administered to the subject in a treatment regimen comprisingintervals of at least 1 hour, or 6 hours, or 12 hours, or 24 hours, or36 hours, or 48 hours between dosages. Administration can be conductedat intervals of at least 72, 96, 120, 168, 192, 216, or 240 hours, orthe equivalent amount of days. The dosage regimen can consist of two ormore different interval sets. For example, a first part of the dosageregimen can be administered to a subject multiple times daily, daily,every other day, or every third day. The dosing regimen can start withdosing the subject every other day, every third day, weekly, biweekly,or monthly. The first part of the dosing regimen can be administered,for example, for up to 30 days, such as 7, 14, 21, or 30 days. Asubsequent second part of the dosing regimen with a different intervaladministration administered weekly, every 14 days, or monthly canoptionally follow, continuing for 4 weeks up to two years or longer,such as 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104 weeks.Alternatively, if the disorder goes into remission or generallyimproves, the dosage may be maintained or kept at lower than maximumamount. If the condition or disorder relapses, the first dosage regimencan be resumed until an improvement is seen, and the second dosingregimen can be implemented again. This cycle can be repeated multipletimes as necessary.

Additional active agents or therapies can be co-administered orotherwise combined with the compounds of the present disclosure.Additional active agents and therapies suitable for use in the methodsof the disclosure include, but are not limited to, compounds used in thetreatment of type-2 diabetes and obesity, such as insulin and insulinanalogues, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-likepeptide-1 analogues, hypoglycemic agents, such as alpha-glucosidaseinhibitors, biguanides, sulfonyl ureas, thiazolidinediones, weight losstherapies, such as appetite suppressing agents, serotonin reuptakeinhibitors, noradrenaline reuptake inhibitors, β3-adrenoceptor agonists,and lipase inhibitors. Compounds used in the treatment of cardiovasculardisease and dysfunction can also be used in the methods disclosure,including, but not limited to, diuretics, angiotensin-converting enzyme(ACE) inhibitors, angiotensin II antagonists, beta-blockers, calciumantagonists, such as nifedipine, HMG-CoA-reductase inhibitors, such asstatins, digoxin, aldosterone antagonists, and organic nitrates. Otherlipid modulating agents including, but not limited to, fibrates and bileacid-binding resins can be used in the methods of the disclosure. Thecompounds of the disclosure can be used with compounds used to assistsmoking cessation including, but not limited to, norepinephrine-dopaminereuptake inhibitors such as bupropion.

Compounds used in the treatment of bone diseases and disorders can beused in the methods of the disclosure. Such compounds include, but arenot limited to, anti-resorptive agents such as bisphosphonates, anabolicagents such as parathyroid hormone, RANKL inhibitors such as denosumab;and estrogen replacement and selective estrogen receptor modulators suchas raloxifene. Compounds used in the treatment of breast cancer, such ascompounds which modulate tubulin polymerization, such as paclitaxel;targeted therapies, such as antibodies against specific cell surfacemarkers on tumor cells, such as antibodies against the HER2 oncoprotein,such as trastuzumab.

Compounds used in the treatment of a disease or disorder with aninflammatory or autoimmune component can be used in the methods of thedisclosure. Such compounds include non-steroidal anti-inflammatory drugs(NSAIDs); disease-modifying anti-rheumatic drugs such asimmunosuppressants; anti-TNF agents, such as infliximab, etanercept, andadalimumab; and anti B-cell therapies, such as rituximab.

Compounds used in the treatment of psychiatric diseases and disorderscan be used in the methods of the disclosure. Such compounds include asGABAA modulators, such as benzodiazepines; 5HT1A receptor agonists, suchas buspirone; beta blockers; antipsychotics, such as dopamine receptorblockers and other drugs which modulate monoamine receptors,transporters or metabolism, such as tricyclic antidepressants, selectiveserotonin reuptake inhibitors, and monoamine oxidase inhibitors;lithium; and anti-epileptic drugs, such as those which block sodiumchannels, those which block T-type calcium channels, or those whichblock GABA transaminase or reuptake, including phenytoin, carbamazepine,valproate and vigabatrin. Compounds used in the treatment of a diseaseor disorder characterized by impairment of memory and/or loss ofcognitive function can also be used in the methods of the disclosure,including, but not limited to such dopamine agonists andanticholinesterases.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising one or more compounds of formulas (I) to (IV) as abovedescribed and one or more pharmaceutically acceptable excipients.

The pharmaceutical compositions can be prepared by any of the methodswell known in the art of pharmacy and drug delivery. In general, methodsof preparing the compositions include the step of bringing the one ormore compounds of formulas (I) to (IV) (active ingredient) intoassociation with a carrier containing one or more accessory ingredients.The pharmaceutical compositions are typically prepared by uniformly andintimately bringing the active ingredient into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation. Thecompositions can be conveniently prepared and/or packaged in unit dosageform.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous solutions and suspensions. Sterileinjectable preparations can be formulated using non-toxicparenterally-acceptable vehicles including water, Ringer's solution, andisotonic sodium chloride solution, and acceptable solvents such as1,3-butanediol. In addition, sterile, fixed oils can be employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

Aqueous suspensions contain the active ingredient in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include, but are not limited to: suspending agents such assodium carboxymethylcellulose, methylcellulose,oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin, polyoxyethylene stearate, and polyethylene sorbitanmonooleate; and preservatives such as ethyl, n-propyl, andp-hydroxybenzoate.

Oily suspensions can be formulated by suspending the active ingredientin a vegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules (suitable for preparation of an aqueoussuspension by the addition of water) can contain the active ingredientin admixture with a dispersing agent, wetting agent, suspending agent,or combinations thereof. Additional excipients can also be present.

The pharmaceutical compositions of the disclosure can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil,for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, such as gum acacia or gum tragacanth;naturally-occurring phospholipids, such as soy lecithin; esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate; and condensation products of said partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate.

Pharmaceutical compositions containing compounds of the disclosure canalso be in a form suitable for oral use. Suitable compositions for oraladministration include, but are not limited to, tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups, elixirs, solutions, buccalpatches, oral gels, chewing gums, chewable tablets, effervescentpowders, and effervescent tablets. Compositions for oral administrationcan be formulated according to any method known to those of skill in theart. Such compositions can contain one or more agents selected fromsweetening agents, flavoring agents, coloring agents, antioxidants, andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations.

Tablets generally contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients, including: inertdiluents, such as cellulose, silicon dioxide, aluminum oxide, calciumcarbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose,calcium phosphate, and sodium phosphate; granulating and disintegratingagents, such as corn starch and alginic acid; binding agents, such aspolyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG),starch, gelatin, and acacia; and lubricating agents such as magnesiumstearate, stearic acid, and talc. The tablets can be uncoated or coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate can beemployed. Tablets can also be coated with a semi-permeable membrane andoptional polymeric osmogents according to known techniques to formosmotic pump compositions for controlled release.

Compositions for oral administration can be formulated as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent (such as calcium carbonate, calcium phosphate, or kaolin), or assoft gelatin capsules wherein the active ingredient is mixed with wateror an oil medium (such as peanut oil, liquid paraffin, or olive oil).

Compounds of the disclosure can also be administered topically as asolution, ointment, cream, gel, suspension, eye-drops, and the like.Still further, transdermal delivery of compounds of the disclosure canbe accomplished by means of iontophoretic patches and the like. Thecompound can also be administered in the form of suppositories forrectal administration of the drug. These compositions can be prepared bymixing the drug with a suitable non-irritating excipient which is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols.

In a related aspect, the disclosure provides a kit having apharmaceutical composition as described above and instructions for use.

The present disclosure provides the following non-limitative examples1-3; each of these examples contains an own general presentation andprior art review, description of material and methods, presentation anddiscussion of results and a list of internal literature references.

Example 1 Analysis of Impurities of Cannabidiol from Hemp. Isolation,Characterization and Synthesis of Cannabidibutol, the Novel CannabidiolButyl Analog Highlights

Cannabidivarin (CBDV) and cannabidibutol (CBDB) are the two majorimpurities in cannabidiol (CBD) extracted from hemp. In this example,CBDB was isolated and fully characterized for the first time. Astereoselective synthesis was carried out and absolute configurationassigned to natural CBD. A match of all properties of isolated CBDB andsynthesized CBDB was obtained. A simple and selective HPLC-UV method wasdeveloped and validated. The method was applied to ten batches ofcommercial CBD marketed by certified companies.

Abstract

Cannabidiol (CBD), one of the two major active principles present inCannabis sativa, is gaining great interest among the scientificcommunity for its pharmaceutical, nutraceutical and cosmeticapplications. CBD can be prepared either by chemical synthesis orextraction from Cannabis sativa (hemp). The latter is more convenientfrom several points of view, including environmental and economic, butmainly for the absence of harmful organic solvents generally employed inthe chemical synthesis. Although CBD produced by hemp extraction is themost widely employed, it carries two major impurities. The first one iscannabidivarin (CBDV), whereas the second one is supposed to be thebutyl analog of CBD with a four-term alkyl side chain. In this work, wereport the isolation by semi-preparative liquid chromatography and theidentification of this second impurity. A comprehensive spectroscopiccharacterization, including NMR, UV, IR, circular dichroism andhigh-resolution mass spectrometry (HRMS), was carried out on thisnatural cannabinoid. In order to determine its absolute configurationand chemical structure, the stereoisomer (1R,6R) of the supposedcannabinoid was synthesized and the physicochemical and spectroscopicproperties, along with the stereochemistry, matched those of the naturalisolated molecule. According to the International Nonproprietary Name,we suggested the name of cannabidibutol (CBDB) for this cannabinoid.Lastly, an HPLC-UV method was developed and validated for thequalitative and quantitative determination of CBDV and CBDB in samplesof CBD extracted from hemp and produced according to Good ManufacturingPractices regulations for pharmaceutical and cosmetic use.

1.1 Introduction

Since its discover by Adams in 1940 [1] and structure elucidation byMechoulam and Shvo in 1963 [2], studies on cannabidiol (CBD, FIG. 1)have undergone profound changes over the time. Initially, it wasconsidered an inactive cannabinoid [3], thus leaving the field to theresearch on the “active” constituent of Cannabis sativa, Δ⁹-tetrahydrocannabinol (Δ⁹-THC). While deepening the knowledge on THC,the studies on CBD were confined to the interaction with the moreinteresting psychotropic isomer [3]. The period of silence on CBDeventually stopped in the early 2000's, when there was a boost in thenumber of publications on this cannabinoid due to the plethora ofpharmacological activities addressed to CBD alone, many of which withtherapeutic potential [3]. More than four hundred papers were publishedlast year on CBD compared to about twenty exactly twenty years ago (fromScopus search “cannabidiol”). In 2018 CBD was approved by FDA for thetreatment of severe forms of infant epilepsy (Lennox-Gastaut syndromeand Dravet syndrome) and it is now commercialized by GW Pharmaceuticals(UK) as a 100 mg/mL oral solution with the name of Epidiolex® [4]. CBDin Epidiolex is extracted from hemp inflorescence and therefore it isproduced according to the Good Manufacturing Practices (GMP). GMP coversall stages of production from the starting materials to the facilities,equipment and processes, but also record keeping, personnelqualifications and training, sanitation and cleanliness. Hence, a drugsubstance that is produced as an Active Pharmaceutical Ingredient (API)should comply to a series of specifications including a detailed reportof the chemical composition. GW Pharmaceuticals clearly indicates thatCBD in Epidiolex is 98% pure, thus it contains some impurities. In itsapplication patent entitled “Use of cannabinoids in the treatment ofepilepsy” [5], GW Pharmaceuticals lists the impurities in the extractedcannabidiol including cannabidiolic acid (CBDA) 0.15% w/w,cannabidivarin (CBDV, FIG. 1) 1% w/w, Δ⁹-THC 0.15% w/w and CBD-C₄(FIG. 1) 0.5% w/w. The latter is intended as4-butyl-5′-methyl-2′-(prop-1-en-2-yl)-1′,2′,3′,4′-tetrahydro-[1,1′-biphenyl]-2,6-diol,which is the analog of CBD with a butyl side chain in place of thepentyl chain on the resorcinol moiety. However, no record of itsphysicochemical and optical characterization can be found in thescientific literature.

The same considerations on GMP are valid for other forms of CBDdifferent from the oil, intended for pharmaceutical use, such as CBDcrystals. CBD in this solid form can be obtained by either extractionfrom Cannabis sativa inflorescence or by a stereoselective synthesis.Natural CBD is generally extracted with organic solvents or bysupercritical carbon dioxide from Cannabis inflorescence, which has beenpreviously decarboxylated since the plant only produces its acidicprecursor CBDA [6]. Alternatively, CBDA can be extracted and thendecarboxylated by heating the extract to get CBD [6, 7]. The extractusually undergoes a “winterization” or dewaxing step in order to removethe waxes and then CBD is purified. Purification can be performed bychromatography or directly crystallized from the winterized extract fromeither pentane or hexane [5, 8]. As an alternative, pure CBD can beproduced via a stereoselective synthesis as reported by Petrzilka et al.[9] and later improved by Baek et al. [10]. The synthetic route involvesthe acid condensation of p-mentha-2,8-dien-1-ol with olivetol. However,beside CBD, as the major product, two main by-products are alwaysobtained, namely a CBD isomer defined as “abnormal CBD” (abn-CBD, 1,FIG. 1) and a CBD with an additional p-mentha1,8-dien-3-yl moiety in 4′position, namely5,5″-dimethyl-6′-pentyl-2,2″-di(prop-1-en-2-yl)-1,1″,2,2″,3,3″,4″,6-octahydro-[1,1′:3′,1″-terphenyl]-2′,4′-diol(2, FIG. 1). Thus, chromatographic purification may be helpful to obtainCBD with a degree of purity suitable for the pharmaceutical use, withconsequent final yield not greater than 60%. Therefore, from botheconomic and ecological point of view, the extraction of CBD fromCannabis inflorescence still remains a suitable process for industrialCBD production.

Notwithstanding the increasing use of CBD in pharmaceutical and cosmeticproducts, there is no related monograph in the official pharmacopoeias.The only official document reporting a protocol for solid or oily CBDformulations is a monograph in the German codex DAC/NRF, which has legalvalue only in Germany [11]. The monograph reports the mainphysicochemical properties, the methods for identification includingthin layer chromatography, and the methods for determining the purityincluding liquid chromatography coupled to UV detection (HPLC-UV). Themonograph also reports the impurities that can be encountered in asample of solid CBD, specifically cannabinol (CBN), Δ⁹-THC and Δ⁹-THC,which together with other minor non specified impurities should be notmore than 0.5% (w/w). The same monograph reports the preparation of anoily formulation of CBD 50 mg/mL in MCT (medium-chained triglycerides).However, the monograph does not mention two of the main impurities thatcan be found in CBD extracted from hemp like CBDV and CBD-C₄. The amountof the two impurities in the final product could be relatively high, upto 1% and 0.5% (w/w) for CBDV and CBD-C₄ respectively [5]. Although GMPprocedures might be slightly different among countries, they allcomplies to general rules. Stacking to ICH guidelines, detection ofimpurities in an Active Pharmaceutical Ingredient (API) is regulated bythe document Q3A(R2), which fixes the threshold for the determination oforganic impurities according to the daily dose of the drug substance.Specifically, an impurity should be identified when present at a levelof 0.10% and qualified at a level of 0.15% in a drug substance with adaily dose below 2 g/day. In a drug substance with a daily dose above 2g/day, an impurity should be identified and qualified when present at alevel of 0.05%. According to these guidelines, both impurities CBDV andCBD-C₄ should be qualified and reported in the certificate of analysisof the CBD product. To this end, suitable analytical methods should beapplied to quantify these two compounds. A certified analytical standardfor CBDV is commercially available and few analytical methods for itsquantitative determination can be found in the literature [7, 12]. Onthe other hand, no analytical standard is available for CBD-C₄ and noanalytical method has been published. Moreover, its identification inCannabis samples or CBD products has been obtained by only means of massspectrometry data. No further characterization has been performed andthe cannabinoid has never been isolated for determination. To the bestof our knowledge and according to exact structure search on SciFinder,the most comprehensive database for chemical literature, only threescientific papers reports the mass spectrometry profile of CBD-C₄ usingeither gas chromatography analysis coupled to mass spectrometry (GC-MS)[13, 14] or Sorptive Tape-like Extraction coupled with Laser DesorptionIonization Mass Spectrometry (STELDI-MS) [15].

In the light of the above, the aim of the present work was to provide afull chemical characterization of CBD-C₄ including high-resolution massspectrometry data (MS and MS/MS in positive and negative ionizationmode) and spectroscopic data [NMR (¹H, ¹³C, COSY, HSQC and HMBC), IR,UV, circular dichroism (CD) and optical rotatory power]. In order todetermine the identity of CBD-C₄, a stereoselective synthesis of thetrans isomer (1R,6R) was carried out and all chemical properties werecompared with those of CBD-C₄ directly isolated from commercial CBDcrystal (extracted from hemp and produced according to GMP regulations)by semi-preparative liquid chromatography. According to theInternational Nonproprietary Name (INN), we suggested for this CBDanalog the name “cannabidibutol” (CBDB). With the pure analyticalstandard of CBDB in hand, a simple and sensitive liquid chromatographymethod coupled to UV detection (HPLC-UV) was developed and validated adhoc in order to quantity both CBDV and CBDB in CBD samples extractedfrom hemp. The method was validated according to ICH guidelines (Q2(R1))in terms of selectivity, linearity, accuracy, precision, dilutionintegrity and stability. Lastly, it was successfully applied to tencommercially available CBD samples marketed by certified companies.

1.2. Experimental 1.2.1 Chemicals and Reagents

Ethanol 96% analytical grade was bought from Carlo Erba (Milan, Italy).Acetonitrile, water and formic acid were all LC-MS grade and purchasedfrom Carlo Erba. Cannabidivarin (CBDV) was purchased as a Cerilliantcertified analytical standard (Sigma-Aldrich, Milan, Italy). Ibuprofen(FIG. 1) was bought from Farmalabor (Canosa di Puglia, Italy). Samplesof pure cannabidiol (extracted from hemp and produced according to GMPregulations) were kindly provided by three companies: RicercheSperimentali Montale S.P.A. (Montale, Italy), Fagron Italia (Bologna,Italy) and CBDepot (Prague, Czech Republic).(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol and5-butylbenzene-1,3-diol were purchased from Combi-blocks (San Diego,Calif., USA) and GreenPharma (Foligno, Italy), respectively. Chemicalsand solvents for the synthesis were reagent grade and used withoutfurther purification.

1.2.2 Synthesis of(1′R,2′R)-5′-methyl-4-pentyl-2′-(prop-1-en-2-yl)-1′,2′,3′,4′-tetrahydro-[1,1′-biphenyl]-2,6-diol,(−)-trans-Cannabidibutol (CBDB)

To a solution of 5-butylbenzene-1,3-diol (83 mg, 0.50 mmol, 1 eq.) andp-toluenesulfonic acid (9 mg, 0.05 mmol, 0.1 eq.) in dry dichloromethane(DCM) (5 mL) at −10° C., under argon atmosphere, a solution of(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol and5-butylbenzene-1,3-diol (76 mg, 0.50 mmol, 1 eq.) in 5 mL of dry DCM wasadded dropwise. The mixture was stirred in the same conditions for 1 hand then quenched with a saturated solution of NaHCO₃ (10 mL). Theresulting mixture was extracted with diethyl ether (2×10 mL). Thecombined organic phases were washed with brine, dried over anhydrousNa₂SO₄ and concentrated. The crude was purified over silica gel(crude:silica gel ratio 1/200, eluent: cyclohexane:DCM 8/2) and all thechromatographic fractions were analyzed by HPLC-UV andUHPLC-HESI-Orbitrap. The fractions containing exclusively CBDB withoutimpurities were collected to give 48 mg of a reddish oil (32% yield,purity >99%).

1.2.3. Isolation of Natural Cannabidibutol

A sample of commercial CBD crystals (1 g) was dissolved in acetonitrile(10 mL) and 0.5 mL aliquots of the solution were injected in asemi-preparative LC system (Octave 10 Semba Bioscience, Madison, USA).The separation was carried out on a fully porous silica stationary phase(Luna 5 μm C18(2) 100 Å, 250×10 mm) (Phenomenex, Bologna, Italy) with amobile phase composed of acetronitrile:0.11% aqueous formic acid 70:30(v/v) at a flow rate of 5 mL/min.

The impurity CBDV eluted at about 12 min, CBDB eluted at about 14 minand CBD eluted between 15 and 20 min. The fractions containing CBDV andCBDB were collected and analyzed by analytical HPLC-UV. The fractionscontaining exclusively CBDB were combined and dried on the rotavapor at70° C. An amount of about 1 mg of CBDB was obtained as a reddish oil.

1.2.4 Chemical and Spectroscopic Characterization of Cannabidibutol

One-dimensional ¹H and ¹³C NMR and two-dimensional NMR (COSY, HSQC andHMBC) were acquired a DPX-600 Avance (Bruker) spectrometer (600.13 MHzfor ¹H NMR and 150.92 MHz for ¹³C NMR). A 10 mg aliquot of syntheticCBDB and 1 mg aliquot of CBDB isolated from CBD were solubilized in 700and 250 μL of CDCl₃ (at 99.96% of deuteration) and placed in a 5 mm and3 mm NMR tube, respectively. All NMR spectra were recorded at 298 K.¹H-NMR were acquired with a spectral width of 13204.2 Hz, a relaxationdelay of 5 s, a pulse width of 11.23 Hz and 16 number of transient.Proton chemical shifts were reported in parts per million (ppm, δ units)and referenced to the solvent residual peaks (CDCl₃ δ=7.26 ppm).Coupling constants are reported in Hertz (Hz). Splitting patterns aredesigned as s, singlet; d, doublet; t, triplet; q, quartet; dd, doubledoublet; m, multiplet; b, broad. ¹³C-NMR were acquired with a spectralwidth of 33.3 kHz, a relaxation delay of 5 s, a pulse width of 10.00 Hzand 128 and 10240 number of transient for syn-CBDB and ext-CBDB,respectively. Carbon chemical shifts were reported in parts per million(ppm, 6 units) and referenced to the solvent residual peaks (CDCl₃δ=77.20 ppm). The COSY were recorded as a 1024×160 matrix with 2transients per t1 increment and processed as a 1024×1024 matrix. TheHSQC spectra were collected as a 1024×256 matrix with 4 transients pert1 increment and processed as a 1024×1024 matrix, and the one-bondheteronuclear coupling value was set to 145 Hz. The HMBC spectra werecollected as a 2048×220 matrix with 8 transients per t1 increment andprocessed as a 2048×1024 matrix, and the long-range coupling value wasset to 8 Hz. IR spectra were recorded at 25° C. on a Perkin-ElmerSpectrum Two ATR-IR, scanning from 450 to 4000 cm⁻¹. Circular dichroism(CD) and UV spectra were acquired on a Jasco (Tokyo, Japan) J-1100spectropolarimeter using a 50 nm/min scanning speed. Quartz cells with a10 mm path length were employed to record spectra in the 400-200 nmrange. Optical rotation (α) was measured with the P-2000 DigitalPolarimeter (cell-length 100 mm, volume 1 mL) from Jasco Europe (Milan,Italy).

1.2.5 HPLC-UV Analyses

High performance liquid chromatography (HPLC) analyses were carried outon an Agilent 1220 Infinity LC System (Waldbronn, Germany), consistingof a vacuum degasser, a binary pump, a manual injector, a columncompartment and a UV detector. The separation of the analytes wasperformed with a Poroshell 120 C18 column (Poroshell 120 SB-C18, 3.0×150mm, 2.7 μm, Agilent, Milan, Italy) eluting a mobile phase composed of0.1% formic acid in both (A) water and (B) acetonitrile (ACN). Anisocratic elution with 70% B was set for 10 minutes, then 95% B waspumped for 5 min and re-equilibration of the column was set for 2 minfor a total run time of 17 min. The flow rate was maintained constant at0.5 mL/min. The loading loop capacity was 6 μL. The loop was washedbefore each run first with 50 μL of ethanol 96% then with 50 μL ofmobile phase. The UV trace was acquired at 228 nm. The analytes peakswere manually integrated using the EZChrom software (AgilentTechnologies), which was employed also for controlling the onlineanalysis.

1.2.6 UHPLC-HESI-Orbitrap Mass Spectrometry Analyses

Ultrahigh-performance liquid chromatography analyses were carried outfor identification and purity test purposes. They were performed on aThermo Fisher Scientific Ultimate 3000 equipped with a vacuum degasser,a binary pump, a thermostated autosampler, a thermostated columncompartment and a Q-Exactive Orbitrap mass spectrometer with a heatedelectrospray ionization (HESI) source. The mass spectrometry parameterswere optimized by direct infusion of the single analytes at theconcentration of 1 μg/mL with a flow rate of 0.1 mL/min through asyringe pump. The HESI parameters were: capillary temperature, 320° C.;vaporizer temperature, 280° C.; electrospray voltage, 4.2 kV (positivemode) and 3.8 kV (negative mode); sheath gas, 55 arbitrary units;auxiliary gas, 30 arbitrary units; S lens RF level, 45. Control ofonline analyses was carried out using Xcalibur 3.0 software (ThermoFisher Scientific, San Jose, Calif., USA). The exact masses of thecompounds were calculated by the Qualbrowser in Xcalibur 3.0 software.The analyses were acquired in full scan data-dependent acquisition(FS-dd-MS²) in positive and negative mode at a resolving power of 70,000FWHM at m/z 200. The other mass analyzer parameters were: scan range,m/z 250-400; AGC, 3e6; injection time, 100 ms; isolation window for thefiltration of the precursor ions, m/z 2. Fragmentation of precursors wasperformed at 30 as normalized collision energy (NCE) by injectingworking mix standard solution at a concentration of 5 μg/L. Detectionwas based on calculated [M+H]⁺ and [M−H]⁻ molecular ions with anaccuracy of 2 ppm, retention time and fragmentation match (fragments m/zand intensity) with pure analytical standards. Analytical selectivitywas assessed by UHPLC-HESI-Orbitrap MS using the same chromatographicconditions employed for the HPLC-UV method except for the differentlength of the column (Poroshell 120 SB-C18, 3.0×100 mm, 2.7 μm, Agilent,Milan, Italy).

1.2.7 Preparation of Standard Solutions

A stock solution of internal standard (ibuprofen 10 mg/mL) was preparedby dissolving 100 mg in 10 mL of acetonitrile. Three serial 1/10dilutions of the internal standard (IS) stock solution were performed toobtain 100 mL of IS working solution with the final concentration of 1μg/mL in ACN.

Stock solution of CBDV (1000 μg/mL in methanol) and CBDB (1000 μg/mL inACN) were properly diluted in the IS working solution to obtaincalibration standard solutions (CS) at the final concentrations of 0.28,1.41, 2.82, 9.40, 28.2 and 56.4 μg/mL for CBDV and 0.12, 0.60, 1.20,4.00, 12.0 and 24.0 μg/mL for CBDB. Independently prepared CBDV and CBDBmix solutions were prepared in IS and used as the low concentrationquality control (LQC) (0.56 μg/mL for CBDV and 0.24 μg/mL for CBDB),medium concentration quality control (MQC) (18.8 μg/mL for CBDV and 8.00μg/mL for CBDB), and high concentration quality control (HQC) (45.1μg/mL for CBDV and 19.2 μg/mL for CBDB) samples. QCs were prepared asfor calibration standards.

1.2.8 Method Validation

In order to demonstrate the reliability and robustness of the method, amethod validation was carried out based on EMA guidelines and inagreement with international guidelines for analytical techniques forthe quality control of pharmaceuticals (ICH guidelines) [16, 17]. Themethod was validated in terms of selectivity, linearity, accuracy,precision, dilution integrity and stability. No matrix effect orrecovery were assessed as the matrix is represented by acetonitrile, forwhich no matrix effect should be encountered as it is present in themobile phase.

Selectivity. Selectivity is performed in order to assess the ability ofthe method to differentiate and quantify the analytes in the presence ofother components in the sample. It was investigated by analyzing blanksamples, samples containing the analytes and authentic standards andcomparing the retention times of potential interfering compounds withthose of reference standards and IS. Identity of the analytes wasassessed by comparing accurate (within 2 ppm error) m/z of [M+H]⁺ and[M−H]⁻ ions and MS/MS spectra of analytical standards with thoseobtained by UHPLC-HESI-Orbitrap for the analytes in authentic samples.

Linearity. Calibration curve was constructed at six non-zero calibrationlevels 0.28, 1.41, 2.82, 9.40, 28.2 and 56.4 μg/mL for CBDV, 0.12, 0.60,1.20, 4.00, 12.0 and 24.0 μg/mL for CBDB, and 1.00 μg/mL for IS. Peakarea ratios of analyte-to-IS were plotted vs actual concentrations.Calibration curve was built at the beginning of each validation day offive consecutive days (n=5). A linear correlation was assumed if thecoefficient of determination (R²) was greater than 0.998 using weighedregression method (1/x²). The back calculated concentrations should bewithin 15% of the nominal concentrations, and within 20% of the lowerlimit of quantification (LLOQ).

Limit of detection (LOD) and limit of quantification (LOQ). Limit ofdetection (LOD) was estimated based on a 3:1 signal-to-noise (S/N)ratio. Standard stock solutions of the analytes were appropriatelydiluted at the levels of their respective estimated LOD values. The LODvalues were then calculated as three times the standard deviation (SD)obtained by repeatedly analyzed standards (n=5). Lower limit ofquantification (LLOQ) was estimated based on a 10:1 S/N ratio andcalculated as ten times the SD of repeatedly analyzed standards. Theupper limit of quantification (ULOQ) was set at 10% above the highestconcentration of the analytes in a concentrated sample of CBD (10mg/mL).

Autosampler carryover. Autosampler carryover was evaluated by runningtwo blank samples after a calibration standard at the ULOQ and after ahigh concentration sample (CBD 10 mg/mL). The carryover should not begreater than 20% of the LOQ for the analytes and 5% for IS.

Accuracy and precision. The precision and accuracy were evaluated atfour levels, LLOQ (0.28 μg/mL for CBDV and 0.12 μg/mL for CBDB), LQC(0.56 μg/mL for CBDV and 0.24 μg/mL for CBDB), MQC (18.8 μg/mL for CBDVand 8.00 μg/mL for CBDB), and HQC (45.1 μg/mL for CBDV and 19.2 μg/mLfor CBDB). Each sample was analyzed in triplicate within a single day todetermine the intra-day precision and accuracy. The replicate analyseswere repeated on freshly prepared standard solutions for five successivedays (n=15) to determine the inter-day precision and accuracy. Theprecision was expressed as coefficient of variation (CV), and theaccuracy was expressed as the percentage of mean calculated compared tonominal concentration.

Dilution integrity. Dilution integrity was carried out using a spikingstandard solution of the analytes prepared by diluting standard stocksolutions to a final concentration that is three times that of the ULOQ(170.4 μg/mL for CBDV and 72.00 μg/mL for CBDB). Dilution integrity wasdemonstrated by diluting the spiking solution in IS to ⅕, 1/10 and 1/20of its original concentration. Five replicates per dilution factor wererun. The concentrations were calculated by applying the dilution factor5, 10 and 20 against freshly prepared calibration curve. Dilutionintegrity is ensured as long as precision and accuracy are <15% and ±15%respectively.

Stability. The short-term stability of the standard analytes wasdetermined for LQC and HQC samples for 24 h at room temperature andunder refrigeration (2-8° C.). The drugs were considered stable if themean concentration (n=3 for each sample) was within +15% of the nominalconcentration.

Preparation and analysis of CBD samples. Authentic CBD samples (APIgrade) were prepared by weighing 10 mg of solid crystals and dissolvingthem in 1 mL of IS working solution. A 100 μL aliquot of the solutionwas diluted with 900 μL of IS working solution to get a CBDconcentration of 1 mg/mL. The HPLC-UV analyses of authentic CBD sampleswere carried out in triplicate employing the validated method describedabove.

1.3. Results and Discussion 1.3.1 Identification of CBD Impurities byMass Spectrometry

CBD samples were analyzed by UHPLC-HESI-Orbitrap, which represents thecutting-edge technology for mass spectrometry allowing for superioraccuracy and precision (below 2 ppm error) in the determination of theexact mass and fragmentation profiles of organic compounds. The mainpeak in the chromatograms (FIG. 2) was identified as CBD by overlap ofthe extracted ion chromatogram (EIC, FIG. 3) and MS/MS spectra of thepure analytical standard of CBD analyzed in the same LC-MS conditions inboth ESI+ and ESI− mode (FIGS. 4(a) and 4(b)). The MS spectrum of thepeak at 4.2 min does not show any other interfering compound. Theprecursor ion [M+H]+ has m/z 315.2314 and the [M−H]− has m/z 313.2179.

As shown in FIG. 3, the peak at 2.6 min (impurity 1) corresponds to theions [M+H]+ with m/z 287.2002 and [M−H]− with m/z 285.1861. Both MS/MSspectra (ESI+ and ESI−) in FIGS. 5(a) and 5(b) present a perfect matchwith those of cannabidivarin (CBDV) analyzed in the same conditions. Thepeak corresponding to this analyte does not present any interferingpeak.

The peak at 3.3 min in FIG. 3 (impurity 2) corresponds to the ions[M+H]+ with m/z 301.2157 and [M−H]− with m/z 299.2016. No interferingpeak was detected at the retention time of this compound. Both MS/MSspectra (ESI+ and ESI−) of impurity 2 are shown in FIGS. 6(a) and 6(b).The elution time is between that of CBDV and CBD, indicating that thepolarity of the compound is greater than CBD and lower than CBDV. Themolecular ions [M+H]+ at m/z 301.2157 and [M−H]− at m/z 299.2016correspond exactly to one methylene unit (—CH₂—) either inserted ontoCBDV or removed from CBD. The fragmentation pattern in both ESI+ andESI-mode corroborates the hypothesis that the difference lies in amethylene unit since both fragments and ion abundance perfectly matchwith those of CBDV and CBD. As highlighted in FIG. 7, which representsthe fragmentation pattern of the three cannabinoids in positiveionization mode, the fragment at m/z 245.1533 in the MS/MS spectrum ofimpurity 2 has the same ionic abundance of the fragments at m/z 259.1960and m/z 153.0909 in the MS/MS spectra of CBD and CBDV respectively.These fragments derive from the loss of four carbon units from theterpene moiety. Similarly, the fragment at m/z 221.1533 for impurity 2derives from the breakage of the terpene group with only four carbonunits left, as it also occurs for the fragments at m/z 235.1690 for CBDand m/z 207.1378 for CBDV. The base peak at m/z 179.1064 also differs byone methylene unit from the base peaks at m/z 193.1222 for CBD and m/z165.0910 for CBDV, and corresponds to the complete loss of the terpenemoiety except for one carbon unit. The fragment at m/z 167.1065 derivesfrom the further loss of the last carbon unit of the terpene moiety togive the protonated molecule of resorcinol with four carbon atoms on thealkyl side chain. The corresponding fragments for CBD and CBDV are atm/z 181.1221 and m/z 153.0909, respectively. The other smaller fragmentsare enclosed in the blue dashed box and correspond to the fragmentationprofile of the terpene moiety, which remains unchanged for all threecannabinoids. In a similar way, the fragmentation pattern of impurity 2in negative ionization mode, shown in FIG. 8, is identical to those ofCBD and CBDV differing by a methylene unit. The base peak at m/z231.1387 in the spectrum of impurity 2 derives from a retro Diels-Alderreaction and loss of a part of the terpene moiety, similarly to thefragments at m/z 245.1546 and m/z 217.1229 for CBD and CBDVrespectively. The other important fragment is at m/z 165.0912 derivingfrom the complete loss of the terpene moiety that leads to the ionizedresorcinol molecule. The corresponding fragments for CBD and CBDV are atm/z 179.1069 and m/z 151.0754 respectively. All fragments and molecularions show the same ionic abundance. These data are in agreement with thehypothesis that impurity 2 is a cannabinoid with molecular formulaC₂₀H₂₈O₂. The data are also in accordance with the high-resolution massspectrometry characterization of CBD, CBDB and CBDV reported in theliterature [18].

1.3.2 Isolation of Natural CBDB

The isolation of impurity 2 from CBD can give an identification. To thisend, a semi-preparative chromatographic method was developed employing asemi-preparative column with a fully porous C18 silica stationary phase(250×10 mm) and a mobile phase composed of ACN and water 70:30 (v/v).This method allowed for the isolation of about 1 mg of impurity 2starting from 1 g of a commercial hemp derived CBD. The amount ofisolated compound resulted sufficient to perform a comprehensivespectroscopic characterization, including NMR, optical rotatory power,UV and CD (Exhibit A—Supporting Information), in order to determine itschemical structure by comparison with the spectroscopic data ofsynthetic CBDB.

1.3.3 Synthesis of CBDB

The synthesis and full spectroscopy characterization of CBDB may nothave been reported in the literature and its existence has beenhypothesized only by means of MS data. In absence of an analyticalstandard or any spectroscopic reference of CBDB that could be used forthe identification of impurity 2, we synthesized and carried out a fullspectroscopic profile of the molecule (−)-trans-CBDB. The latter wasprepared by Friedel-Craft allylation of 5-butylbenzene-1,3-diol with(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using pTSA ascatalyst as reported in Scheme 1.

1.3.4 Spectroscopic Characterization of Syn-CBDB and Ext-CBDB

The chemical identity of synthetic CBDB and its ¹H assignments wereachieved by analysing ¹H-NMR, ¹³C-NMR and COSY spectra. The protonatedcarbon atoms were assigned by analysis of the HSQC spectra, and thequaternary carbons were assigned based on HMBC spectra. Table 1 and FIG.9 show the ¹H and ¹³C NMR complete assignments for CBDB, determining thechemical structure of the new synthesized cannabinoid. However, sincethe molecule of CBDB possesses two stereocenters, it could exist as fourpossible stereoisomers. The synthetic procedure adopted may have lead tothe stereoselective synthesis of (−)-trans-CBDB, and the exact positionof the double bonds and the configuration of carbon 1 and 6 were fullyinvestigated and determined by NMR.

Indeed, even though the NMR is not able to discriminate between a coupleof enantiomers, a slight shift of the proton signals could be detectedamong a couple of diastereomers (i.e. cis/trans isomers). Because CBDBand CBD differ only for a methylene in the alkyl chain on the resorcinolmoiety, no significative difference in the proton chemical shifts of theterpene moiety may exist among the two molecules. Thus, in order todefine first a cis or trans configuration for the synthetic CBDB, wecompared its ¹H-NMR with the spectra of (−)-trans-CBD [19] and(−)-cis-CBD [9]. As reported in Table 2, it is possible to observe aclose match between the H-1, H-2 and H-6 signals of synthetic CBDB andthe corresponding signals of (−)-trans-CBD. In contrast, cis-CBDpresents a shielding for H-1 and H-2 (from 3.81-3.84 to 3.35-3.75 andfrom 5.54 to 5.40, respectively) and a deshielding for H-6 (from 2.38 to2.90). These outcomes suggested, therefore, a trans configuration forthe synthetic CBDB. However, as trans-CBDB can exist as an enantiomericcouple (1R,6R) or (1S,6S), in order to establish the absoluteconfiguration of C-1 and C-6, the optical rotatory power was determined,resulting in an [α]_(D) ²⁰=−121° (c. 1.6, ACN). This value is in linewith the [α]_(D) ²⁰=−125° of (−)-trans-CBD, allowing the assignment ofthe (1R,6R) absolute configuration and the identification of thesynthetic CBDB as (−)-(1R,6R)-CBDB.

Based on the chemical structure and stereochemistry of the synthesized(−)-trans-CBDB, the latter was used as reference compound to determinethe identity of the impurity 2. The NMR spectra, UPLC retention time,m/z precursor ions ([M+H]⁺ and [M−H]⁻), MS, UV and CD spectra of bothsynthetic CBDB and impurity 2 (isolated natural CBDB) were compared. Asreported in FIG. 10, a perfect superimposition between the spectroscopicdata of the two molecules was observed. Moreover, the specific rotatorypower of isolated CBDB resulted in an [α]_(D) ²⁰=−116° (c. 0.5, ACN)comparable to the value obtained for the synthetic CBDB. Therefore,based on these considerations, we can state that the impurity 2 presentin the CBD extracted from hemp inflorescence is (−)-trans-CBDB.

1.3.5 HPLC-UV Method Development and Validation

Our research group has recently published several works on thedevelopment and optimization of analytical methods for the separation ofcannabinoids by LC-UV and LC-MS [7, 12, 18, 20-23]. In order to achievethe optimal resolution of the analytes CBDV and CBDB, a core shellcolumn (Poroshell 120 SB-C18, 3.0×150 mm, 2.7 μm) was employed. Themobile phase consisted of 0.1% aqueous formic acid and acetonitrile(30:70, v/v), which allowed for the separation of the analytes within 7min and of the main peak of CBD within 10 min. FIG. S2 in the ExhibitA—Supplementary Material shows the LC-UV chromatograms of a standardmixture at different concentration levels and an example of an authenticCBD sample. In order to assess the reliability and robustness of themethod, a validation according to EMA and ICH guidelines was performedin terms of linearity, selectivity, carryover, accuracy, precision,dilution integrity and stability [16, 17], and the results are describedbelow. The tables of the validation results are reported in the ExhibitA—Supplementary Material.

Linearity. Linearity was assessed for CBDV and CBDB in the ranges0.28-56.4 μg/mL for CBDV and 0.12-24.0 μg/mL for CBDB. Theconcentration-response relationship was based on a weighted linearregression (1/x²) in order compensate for the error at lowconcentrations considering the high dynamic range covered by thecalibration curve. The coefficient of determination (R²) was found above0.999 for both analytes CBDV and CBDB, thus the correlation ofconcentration vs UV response was considered linear in the specifiedrange (Table S1).

Limit of quantification (LOQ) and limit of detection (LOD). LOD wascalculated as described in the methods section and was found 0.10 μg/mLfor CBDV and 0.04 μg/mL for CBDB. The LLOQ, which is also the lowestcalibration point was 0.28 μg/mL for CBDV and 0.12 μg/mL for CBDB. TheULOQ was set 10% above the highest concentration found for the analytesby injecting a high concentration CBD sample (10 mg/mL). Consideringthat the highest concentration of CBDV and CBDB in that concentrated CBDsample was 51.0 and 21.0 μg/mL, respectively, the ULOQ was set at 56.4and 24.0 μg/mL for CBDV and CBDB, respectively.

Autosampler carryover. The analyses performed to assess the autosamplercarryover indicated that the peak area corresponding to the analytes wasnot greater than 17% of the LLOQ and it was totally absent for the IS,thus ensuring good reliability of the quantification of the analytes.

Accuracy and precision (CV). Intra-day accuracy ranged from 98.23 to104.9% for CBDV and from 100.3 to 105.5% for CBDB. Intra-day precision,expressed as coefficient of variation (CV), was found in the range0.98-2.25% for CBDV and in the range 1.62-12.0% for CBDB. Inter-dayaccuracy ranged from 102.0 to 109.0% for CBDV and from 91.67 to 102.0%for CBDB. Inter-day CV was in the range 0.96-2.76% for CBDV and in therange 2.37-9.14% for CBDB. The acceptance criteria of EMA guidelines areestablished in the range 85-115% for accuracy and below 15% for CV(Table S2). Given the data above, it can be inferred that the developedmethod is accurate and precise.

Dilution integrity. For dilution integrity accuracy and CV wereevaluated across five analyses of a highly concentrated standard mixtureof CBDV and CBDB prepared with a concentration three times higher thanthe ULOQ and diluted to ⅕, 1/10 and 1/20. The accuracies were found inthe range 96.72-99.53% for CBDV and in the range 93.47-96.00% for CBDB.The CV was below 3% for both analytes (Table S3). Since accuracy andprecision were within ±15% of the nominal concentration, the results metthe EMA acceptance criteria, ensuring that samples with concentrationsgreater than the ULOQ could be diluted and quantified with a good levelof confidence. Moreover, this suggests that the calibration range couldbe extended above the ULOQ set in this method.

Stability. The stability of the analytes was evaluated at twoconcentration levels, LQC and HQC, at two different temperatures, roomtemperature (25° C.) and under refrigeration (2-8° C.), in a timeinterval of 24 hours. The analytes were found stable in both conditionsas the calculated concentration was within 5% of the nominalconcentration using a freshly prepared calibration curve (Table S4).

1.3.6 Analyses of Authentic CBD Samples

Authentic CBD samples (1 mg/mL), coming from different batches producedaccording to GMP regulations by hemp extraction and provided by threemanufacturers, were analyzed according to the developed HPLC-UV methodas described in the experimental section. The results obtained for theconcentrations of CBDV and CBDB present in the samples are reported inTable 3. These impurities were present in amounts lower than 0.5%, inparticular CBDV was found in the range 0.07-0.41% and CBDB was in therange 0.08-0.19%. The values are extremely variable across the tensamples, most likely because both hemp variety and industrial productmanufacturing affect the amount of impurities eventually present in thefinal product. CBDV has already been detected in several hemp varietiesin extremely variable concentrations [7, 24-27]. CBDB was also detectedin some hemp varieties but its concentrations have never been determineddue to the lack of the corresponding analytical standard [13, 14, 28].It is reasonable to assume that the concentrations of CBDV and CBDB inCBD samples are a mirror of the concentrations of these analytes in theoriginal plant material from which CBD is extracted. Moreover, since CBDis generally extracted from hemp by crystallization without furtherpurification, the structural similarity of CBDV, CBDB and CBD leads to aco-crystallization of the three compounds. Chromatography would be theonly means that can allow to remove such impurities and obtain a 99.99%pure CBD. However, this involves the use of organic solvents, which inturn can be found as a residual impurity in the final product, thusrepresenting a detrimental method from an ecological point of view.

1.4 Conclusions

One of the major impurities of CBD extracted from hemp, cannabidibutol(CBDB), was fully characterized for the first time. A stereoselectivesynthesis was developed in order to determine its identity andstereochemistry. This allowed to obtain for the first time the authenticanalytical standard, which was employed for the development andvalidation of an HPLC-UV method for its qualitative and quantitativedetermination in commercial samples of CBD produced according to GMPregulations. Such standard and analytical method may bridge the gap forpharmaceutical and cosmetic industries that produce CBD. Moreover,although the monograph of CBD in the German DAC code does not mentionthe presence of either CBDV or CBDB, the latter are the two majorimpurities in CBD extracted and crystallized from hemp. These twoimpurities should be included along with the analytical method for theirdetermination in a desirable monograph on CBD of an officialpharmacopoeia. Considering that CBDB is present in hemp, as reported byfew articles, it may be found also in the acidic form ascannabidibutolic acid (CBDBA), without ruling out the presence of thecorresponding ring-closed isomer, tetrahydrocannabutolic acid (THCBA),and of the neutral derivative tetrahydrocannabutol (THCB). The ongoingresearch in our laboratory aims at identifying these molecules indifferent Cannabis varieties.

5. Tables

TABLE 1 ¹H and ¹³NMR assignments (δ) of CBDB^([a]).

Position ¹H-NMR^([b]) ¹³C-NMR 1 3.82-3.86 (m)  37.50 2  5.57 (s) 124.293 140.25  4a 2.08-2.12 (m)  30.59  4b 2.22-2.24 (m) 5 1.76-1.85 (m) 28.60 6  2.40 (dt)  46.32 7 1.79 (s)  23.87 8 149.62  9a 4.56 (s)111.01  9b 4.67 (s) 10 1.65 (s)  20.76  1′ 154.01  2′ 113.92  3′ 156.29 4′  6.17 (bs) 108.17  5′ 143.19  6′  6.28 (bs) 110.02  7′  4.58 (bs) 8′ 5.97 (s)  1″ 2.45 (t)  35.37  2″ 1.52-1.57(m)  33.29  3″  1.32 (sxt) 22.51  4″ 0.90 (t)   14.15 ^([a])NMR spectra were recorded in CDC1₃99.9% of deuteration on a Bruker 600 spectrometer with ¹H at 600 MHz and¹³C at 151 MHz. Chemical shifts are reported in parts per million (ppm,δ units). Proton chemical shifts were referenced to the solvent residualpeak of CDC1₃ (7.26 ppm). ^([b])Splitting patterns are designed as s,singlet; t, triplet; sxt, sextet; dt, double triplet; m, multiplet; b,broad.

TABLE 2 Comparison between the chemical shift of the proton signals ofthe terpene moiety among trans-CBDB, trans-CBD and cis-CBD. H-NMR (δ)Position trans-CBDB (-)-trans-CBD (-)-cis-CBD 1 3.82-3.86 3.81-3.843.75-3.35 2 5.57 5.54 5.40 6 2.40 2.38 2.90

TABLE 3 Analysis of authentic CBD samples produced according to GMPregulations. The values are expressed as mean percentage (w/w) of threeanalyses (n = 3, standard deviation is not indicated as it was lowerthan 0.0001 for all samples). Sample CBDV (%) CBDB (%) CBD-1 0.07 0.08CBD-2 0.15 0.10 CBD-3 0.34 0.16 CBD-4 0.25 0.13 CBD-5 0.19 0.17 CBD-60.17 0.11 CBD-7 0.33 0.16 CBD-8 0.41 0.19 CBD-9 0.33 0.23 CBD-10 0.270.22

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Example 2 Isolation of a High Affinity Cannabinoid for Human CB1Receptor from a Medicinal Cannabis Variety: Δ⁹-Tetrahydrocannabutol, theButyl Homologue of Δ⁹-Tetrahydrocannabinol

Abstract: The butyl homologues of Δ⁹-tetrahydrocannabinol,Δ⁹-tetrahydrocannabutol (Δ⁹-THCB), and of cannabidiol, cannabidibutol(CBDB), were isolated from a medicinal Cannabis variety (FM2)inflorescence. A comprehensive spectroscopic characterization, includingNMR, UV, IR, circular dichroism and high-resolution mass spectrometry,were carried out on both cannabinoids. The chemical structure andabsolute configuration of the isolated cannabinoids were determined bymatch with the spectroscopic data of the respective compounds obtainedby stereoselective synthesis. The butyl homologue of Δ⁹-THC, Δ⁹-THCB,showed an affinity for the human CB1 (K_(i)=15 nM) and CB2 receptors(K_(i)=51 nM) comparable to that of (−)-trans-Δ⁹-THC. Docking studiessuggested the key bonds responsible for THC-like binding affinity forCB1. The formalin test in vivo was performed on the two compounds inorder to reveal possible analgesic and anti-inflammatory properties. Thetetrad test in mice showed a partial agonistic activity of Δ⁹-THCBtowards CB1 receptor.

2.1 Introduction

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) are two activeprinciples of Cannabis sativa L. (FIG. 11), a plant used for a plethoraof medicinal and nutraceutical properties.^(1, 2) The use of Cannabisextracts has been approved by several countries for the treatment of aseries of pathological conditions, such as chronic pain and painassociated with multiple sclerosis and spinal cord injury; nausea andvomiting caused by chemotherapy, radiotherapy, HIV therapies; lack ofappetite in patients with cachexia, anorexia, chemotherapy or those withAIDS and anorexia nervosa; glaucoma; Tourette syndrome.³⁻⁶ Δ⁹-THC andCBD are present in different ratios of concentrations according to theCannabis variety. Indeed, specific varieties have been selected toproduce predominantly THC leaving CBD levels below 1% (w/w), and viceversa. Alternatively, some pathologies may require the treatment withCannabis varieties containing balanced levels of bothcannabinoids.^(7, 8) To this end, some companies have developed a numberof certified varieties with standardized concentrations of THC and CBD.For example, in The Netherlands the company Bedrocan produces fivevarieties: Bedrocan (22% THC, <1% CBD), Bedica (14% THC, <1% CBD,indica), Bedrobinol (14% THC, <1% CBD, sativa), Bediol (6.3% THC, 8%CBD), and Bedrolite (<1% THC, 9% CBD). In Italy, the MilitaryChemical-Pharmaceutical Institute currently produces two varieties namedFM1 (13-20% THC, <1% CBD) and FM2 (5-8% THC, 7-12% CBD). Canada hasseveral licensed producers of medicinal Cannabis, including CanopyGrowth Corporation, Aurora Cannabis Enterprises Inc., MedRelief Corp,Tilray and many others; each of them has developed its own varieties.Different variety means different concentrations of the activeprinciples, which includes mainly THC and CBD, but also othercannabinoids with lower concentrations but endowed of severalpharmacological properties. Many reports are devoted to thedetermination of cannabinoids in different Cannabis varieties.⁹⁻¹⁶Metabolomics is a useful tool that allows for the detection andidentification of a great number of compoundssimultaneously.^(9, 17, 18) Metabolomics has been successfully appliedin the past fifteen years in the field of plant science and has led tohuge progresses in the knowledge of Cannabis constituents.^(9, 19-24)Our research group has devoted great effort to the development ofsuitable analytical methods for the qualitative and quantitativedetermination of cannabinoids in different matrices, spanning medicinalpreparations, food products and animal tissues.^(14, 22-26) Our mostrecent work has regarded the development of an analytical method for thedetermination of a novel homologue of CBD with a 4-term alkyl side chaininstead of a pentyl chain named cannabidibutol(4-butyl-5′-methyl-2′-(prop-1-en-2-yl)-1′,2′,3′,4′-tetrahydro-[1,1′-biphenyl]-2,6-diol,CBDB).²⁷, 28 Although CBDB (FIG. 11) has been previously detected insome Cannabis varieties by metabolomics-based techniques,^(29, 30) onlyrecently our research group fully characterized this cannabinoid for thefirst time.^(27,28) CBDB was isolated from CBD samples extracted fromindustrial hemp, suggesting that it should be present in the originalplant material. As for the other cannabinoids, Cannabis plant mayproduce the acidic form of CBDB that is cannabidibutolic acid (CBDBA),which is then converted to its neutral counterpart via a decarboxylationreaction triggered by heat (FIG. 11).³¹ THC rich Cannabis varietiesshould contain, although in relatively small amount, the ring closedhomologues of CBDB and CBDBA, Δ⁹-tetrahydrocannabutol (Δ⁹-THCB) andtetrahydrocannabutolic acid (THCBA) (FIG. 11). Before this work, Δ⁹-THCBhas been already detected in Cannabis plant material, along with CBDB,but it has been characterized only by its mass spectrometricprofile.^(29, 32, 33)

The metabolomics analysis of ethanolic extracts of different varietiesof the species Cannabis sativa showed that the FM2 variety presented thehighest amounts of butyl derivatives of acidic cannabinoids,particularly higher than the corresponding Dutch strain Bediol (byBedrocan, The Netherlands). For this reason, FM2 was selected for theisolation of CBD and THC butyl homologues. Chemical and spectroscopicproperties of isolated neutral compounds were compared to in housesynthesized standards. The latter were then employed for thesemi-quantification of CBDB and Δ⁹-THCB in authentic FM2 samples.Finally, we tested these compounds in a widely used model of acuteinflammatory pain induced by intra-paw formalin injection in order toinvestigate possible anti-inflammatory and analgesic properties of thecompounds under investigation.

2.2. Results and Discussion 2.2.1 Characterization of FM2 CannabisVariety by UHPLC-HESI-Orbitrap

The Italian Cannabis variety FM2 is characterized by a balanced highamount of both THC and CBD. Although these two are the most abundantcannabinoids and their content has been standardized in order to reducechemical variability of medicinal preparations, other minor cannabinoidsare present in the inflorescence. One of the most useful analyticaltools for the characterization of an FM2 ethanol extract is representedby the metabolomics approach with the aid of the Orbitrap technology.The accuracy and precision of the high-resolution mass spectrometry(HRMS) is interfaced to a liquid chromatography platform that operateswith a sub-3 μm fused-core particle stationary phase, which ensures highefficiency and resolution power with modest backpressures.^(14, 22-25, 34, 35) The analysis of the FM2 ethanol extractshowed the presence of high amounts of the acidic precursors of CBD andTHC, CBDA and THCA, and trace amounts of the neutral species since nodecarboxylation process occurred during either the extraction procedureor the storage (FIG. 12). Moreover, CBDA and THCA homologues were alsodetected in the chromatogram. Specifically, the peak of CBDA (17.4 min)was preceded by the peaks of its propyl and butyl homologues,cannabidivarinic acid (CBDVA) and CBDBA, which eluted at 16.1 min and16.8 min respectively. Similarly, the peak of THCA (20.7 min) waspreceded by the peaks of tetrahydrocannabivarinic acid (THCVA) and THCBAthat eluted at 19.0 min and 19.5 min respectively. The identification ofTHCA, CBDA, THCVA and CBDVA was determined by the analysis of thecorresponding analytical standards in the same conditions. However, dueto the lack of the analytical standards of CBDBA and THCBA, thestructural identification was based on exact mass (Δppm<2) andhigh-resolution MS/MS spectrum (FIGS. S1-6, Exhibit B—SupportingInformation). Moreover, given the general difficulty in isolating theacidic forms of cannabinoids, the best way to determine the structuralidentification is to decarboxylate the native plant material by heat,thus providing the corresponding neutral forms. The decarboxylatedinflorescence was extracted as for the native one and subject toUHPLC-HESI-Orbitrap analysis. The peak areas of the acidic species areclearly lower after decarboxylation, while the peak areas of the neutralspecies showed an increase as a consequence of the conversion of theirprecursors.

The interpretation of the high-resolution fragmentation spectradetermined a four-carbon side chain on the resorcinyl moiety. Theprecursor ions of the butyl homologues differed by a methylene unit fromthe pentyl and propyl homologues, as well as the chromatographicretention time, suggested that the lipophilicity of the butyl homologuesis intermediate between the propyl and the pentyl ones. CBDA and THCAhomologues can be better distinguished by their fragmentation profile innegative ionization mode rather than the one in positive mode. Inparticular, THCA homologues are generally more poorly fragmented thanCBDA homologues in negative ionization mode (FIGS. S1 and S2respectively, Exhibit B—Supporting Information). HRMS spectra of CBDBAend THCBA in positive ionization mode were also analyzed and comparedwith their propyl and pentyl homologues (FIGS. S3 and S4, respectively,Exhibit B—Supporting Information). Except for the relative abundance(RA) of the fragments, no marked differences could be noted between CBDand THC homologues of the same series (e.g. CBDA and THCA, CBDBA andTHCBA, CBDVA and THCVA).

The chromatogram of the decarboxylated extract showed an increase in thepeak area of the neutral species. As shown in FIG. 12, the order ofelution was: cannabidivarin (CBDV) (16.4 min), CBDB (17.1 min), CBD(17.8 min), Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV) (18.0 min), Δ⁹-THCB(19.0 min) and Δ⁹-THC (19.8 min). The identity of CBDV, CBD, Δ⁹-THCV andΔ⁹-THC was assigned by analysis of the pure analytical standards in thesame LC-MS conditions. The analysis of the MS and MS/MS spectra inpositive and negative ionization mode of the neutral derivative of CBDBAindicated a perfect match with those of the authentic standard of CBDBsynthesized in our previous work.²⁷ On the other hand, the neutralderivative of THCBA was identified by comparison with its pentyl andpropyl counterpart as a pure analytical standard is not available. TheMS/MS spectrum of Δ⁹-THCB in positive ionization mode isundistinguishable from that of its CBD counterpart,²⁷ but different innegative ionization mode. The few fragments present in thehigh-resolution spectrum of Δ⁹-THCB in negative mode have low abundancecompared to those in the spectrum of CBDB. The spectrum of Δ⁹-THCB inpositive mode is easily comparable to those of Δ⁹-THCV and Δ⁹-THC interms of m/z and RA of the fragments (FIG. S6, Exhibit B—SupportingInformation).

2.2.2 Isolation of Natural CBDB and Δ⁹-THCB

In order to provide an identification of CBDB and THCB, we isolated thetwo compounds from decarboxylated FM2 inflorescence by means ofsemi-preparative liquid chromatography. About 1 mg of pure CBDB and 1 mgof pure Δ⁹-THCB were recovered starting from 4 g of decarboxylated plantmaterial. Both compounds were fully characterized by NMR, IR, UV, CD,optical rotation and HRMS. The properties of isolated CBDB were comparedto those of the previously synthesized analytical standard,^(27,28) thusdetermining its identity. On the other hand, the pure analyticalstandard of Δ⁹-THCB has never been available. To this end, we developeda stereoselective synthesis of the (6aR,10aR) isomer and carried out theassessment of the structural identity and absolute configuration of thetwo chiral centers of Δ⁹-THCB present in the native Cannabisinflorescence.

2.2.3 Stereoselective Synthesis and Spectroscopic Characterization of(−)-Trans-Δ⁹-THCB and Matching with Extracted Δ⁹-THCB

The stereoselective synthesis of (−)-trans-THCB was initially performedby direct condensation of 5-butylbenzene-1,3-diol with(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol using BF₃.Et₂O orZnCl₂ as Lewis' acid, as already reported in literature for thesynthesis of the homologue Δ⁹-THC.³⁶⁻³⁹ Unfortunately, with thesesubstrates, this procedure led to a complex mixture of isomers of CBDBand Δ⁹-THCB resulting in an arduous and low-yield isolation of(−)-trans-Δ⁹-THCB by standard chromatographic techniques. Therefore,with the aim to obtain high amount of pure (−)-trans-Δ⁹-THCB, thesynthetic approach described in Scheme 1 was followed. The Friedel-Craftallylation of 5-butylbenzene-1,3-diol with(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using pTSA ascatalyst, and for a reaction time no longer than 60 minutes, allows toobtain selectively (−)-trans-CBDB, as already reported in our previouswork.²⁷ In contrast, longer reaction times allow the reaction to proceedwith the cyclization of CBDB to Δ⁹-THCB (not isolable at this stage) andthen, quantitatively, to the more thermodynamically stable isomers(−)-trans-Δ⁸-THCB. By addition of hydrochloric acid to the Δ⁸ doublebond, using ZnCl₂ as catalyst, (−)-trans-Δ⁸-THCB was quantitativelyconverted to (−)-trans-HCl-THCB. Finally, elimination of hydrochloricacid on HCl-THCB, performed with potassium t-amylate as base, occurredselectively in position 2 of the terpene moiety, leading to(−)-trans-Δ⁹-THCB in 91% of yield (Scheme 1).

The chemical identity of synthetic (−)-trans-Δ⁹-THCB and its ¹H and ¹³Cassignments were achieved by analyzing ¹H and ¹³C NMR spectra. Table 4and FIGS. S1-2 (Exhibit B—Supporting Information) show the ¹H and ¹³CNMR complete assignments for (−)-trans-Δ⁹-THCB. The synthetic protocoladopted goes through the synthesis of (−)-trans-CBDB, whosestereochemistry was fully investigated and determined in our previouswork²⁷ and subsequent isomerization to Δ⁹-THCB. Although the syntheticconditions may not affect the configuration of the stereocenters of thefinal (−)-trans-Δ⁹-THCB,^(38, 40) we determined the exact position ofthe double bonds and the cis/trans configuration of the terpene ring.Since (−)-trans-Δ⁹-THC with (−)-trans-Δ⁹-THCB differ only by a methylenein the alkyl chain on the resorcinol moiety, no significant differencein the proton chemical shifts of the terpene moiety may exist betweenthe two molecules. The NMR spectra of (−)-trans-Δ⁹-THC and(−)-trans-Δ⁹-THCB were acquired in the same solvent.⁴¹ A near-perfectmatch in the chemical shift of the two molecules was observed, thusdetermining the chemical structure of the new synthesized cannabinoidand in particular the Δ⁹ position of the double bond and the transconfiguration of the dihydro-pyran ring. In addition, the exactconfiguration of carbon 1 and 6 of the synthesized Δ⁹-THCB wasdetermined by the optical rotatory power. (−)-trans-Δ⁹-THCB showed an[α]_(D) ²⁰=−143° in acetonitrile comparable with the [α]_(D) ²⁰=−152° inethanol of (−)-trans-Δ⁹-THC.⁴² Since both molecules resulted levogyre,this suggests that the configuration at carbon 1 and 6 is the same forΔ⁹-THC and Δ⁹-THCB, namely (1R, 6R).

TABLE 4 ¹H and ¹³ NMR Assignments (δ) of (-)-trans-Δ⁹-THCB and(-)-trans-Δ⁹-THC in CDCl₃ ^(a) (-)-trans-Δ⁹-THCB (-)-trans-Δ⁹-THC⁴¹position^(b) ¹H NMR ¹³C NMR ¹H NMR ¹³C NMR  1 — 154.91 — 154.7  2 6.14(1H, s) 107.69 6.14 (1H, d, J = 1.6 Hz) 107.5  3 — 142.90 — 142.8  46.27 (1H, s) 109.18 6.27 (1H, d, J = 1.6 Hz) 110.1  4a — 154.33 — 154.2 6 —  77.35 —  76.7  6a 1.66-1.72 (1H, m)  45.95 1.69 (1H, m)  45.8  7a-1.89-1.93 (1H, m)  25.16 b-1.90 (1H, m)  25.0   b-1.37-1.43 (1H, m)b-1.40 (1H, m)  8 2.15-2.17 (2H, m)  31.31 2.16 (2H, m)  31.2  9 —134.54 — 134.3 10 6.29 (1H, s) 123.88 6.31 (1H, q, J = 1.6 Hz)  13.7 10a3.20 (1H, d, J = 12.0 Hz)  33.72 3.20 (1H, d, J = 10.9 Hz)  33.6 10b —110.24 — 110.8 11 1.68 (3H, s)  23.50 1.68 (3H, s)  23.4 12 1.41 (3H, s) 27.71 1.41 (3H, s)  27.6 13 1.09 (3H, s)  19.41 1.09 (3H, s)  19.3 OH4.64 (1H, bs) — 4.87 (1H, s) —  1′ 2.45 (2H, t, J = 8.0 Hz)  35.31 2.42(2H, td, J = 7.3, 1.6 Hz)  35.5  2′ 1.56 (2H, qnt, J = 8.0 Hz)  33.251.55 (2H, q, J = 7.8 Hz)  30.6  3′ 1.36 (2H, sxt, J = 8.0 Hz)  22.481.29 (4H, m)  31.5 {close oversize brace}  4′ 0.90 (3H, t, J = 8.0 Hz) 14.09  22.5  5′ N/A N/A 0.87 (3H, t, J = 7.0 Hz)  14.0 ^(a)Chemicalshift, in ppm, are referenced to the chloroform residual signal (7.26ppm for ¹H and 77.20 ppm for ¹³C). Coupling constants are reported inhertz (Hz). Splitting patterns are designed as s, singlet; d, doublet;t, triplet; qnt, quintet; sxt, sextet; m, multiplet; b, broad.^(b)According to dibenzopyran numbering. N/A. Not applicable

Based on the chemical structure and stereochemistry of syntheticΔ⁹-THCB, the latter was used as reference compound to determine theidentity of the (−)-trans-Δ⁹-THCB identified in FM2 Cannabis variety.The UHPLC retention time, m/z precursor ions ([M+H]⁺ and [M−H]⁻) andfragmentation spectra of both molecules showed a perfect match.Moreover, the comparison of ¹H and ¹³C NMR, UV and CD spectra of bothsynthetic Δ⁹-THCB and isolated Δ⁹-THCB resulted in a perfect overlap(FIGS. S17-S18, Exhibit B—Supporting Information). Therefore, based onthese considerations, we can state that the new identified cannabinoidpresent in the extracted from FM2 hemp inflorescence is the(−)-trans-Δ⁹-THCB.

2.2.4 Binding Affinity at Human CB1 and CB2 Receptors

The binding affinity of (−)-trans-Δ⁹-THCB against purified human CB1 andCB2 receptors was determined in a radioligand binding assay. Thecapability of the ligand to displace radiolabeled [3H]CP55940 or [³H]WIN55212-2 from CB1 and CB2 receptors, respectively, was measured at tenconcentration ranging from 1 nM to 30 uM and the IC₅₀ and K_(i) valueswere calculated (Table 5 and Exhibit B—Supporting Information). CP55940(CB1 IC₅₀=1.7 nM, CB1 K_(i)=0.93 nM) and WIN 55212-2 (CB2 IC₅₀=2.7 nM,CB2 K_(i)=1.7 nM) were used as reference compounds. (−)-trans-Δ⁹-THCBbinds with high affinity to both human CB1 and CB2 receptors with aK_(i) of 15 and 51 nM, respectively. Comparing these data with thosereported in the literature, (−)-trans-Δ⁹-THCB resulted three times moreactive than (−)-trans-Δ⁹-THC (K_(i)=40 nM) and five times more activethan (−)-trans-Δ⁹-THCV (K_(i) of 75.4 nM) against CB1 receptor, whereasno significative difference in biding affinity were observed against CB2receptor for the three cannabinoids.⁴³

TABLE 5 Binding Affinity (IC₅₀ and K_(i)) of (-)-trans-Δ⁹- THCB at humanCB1 and CB2 Receptors. hCB1 hCB2 IC₅₀ in nM K_(i) in nM IC₅₀ in nM K_(i)in nM (-)-trans-Δ⁹-THCB 28 15 79 51 CP 55940 1.7 0.93 — — WIN 55212-2 —— 2.7 1.7 SD is within ± 10% of the value

In order to explore the binding affinity against CB1 receptor, dockingsimulation for the three THC homologues was performed. The x-raystructure of the active conformation of CB1 receptor in complex with theagonist AM11542 (PDB ID: 5XRA) was used as reference for docking sincemarked structural changes in the orthosteric ligand-binding site wereobserved with the respect of the inactive conformation.⁴⁴⁻⁴⁶ As reportedin FIG. 13A-C, the three tetrahydrocannabinols (THC, THCV and THCB)exhibited similar binding poses in the orthosteric ligand-binding site.In particular, no significative differences were observed for the poseof the tetrahydrobenzo[c]chromene core located in the main hydrophobicsite. The aromatic ring of resorcinol is involved in an edge-to-face7L-7L interaction with Phe170 and Phe268, whereas the hydroxyl group isengaged in a H-bond with Ser383 (FIG. 13A-C). The main differencebetween the three ligands is observed in the position of the aliphaticside chain. Indeed, the pentyl side chain of Δ⁹-THC protrudes into thelong tunnel formed by helices III, V and VI, undergoing hydrophobicinteractions with Leu193, Val196, Tyr275, Leu276, Trp279 and Met363. Incontrast, the propyl chain of Δ⁹-THCV is located in a small hydrophobicsub-pocket located near the entrance of the tunnel and delimited byPhe170, Phe200, Leu387, Met363, Leu359 and Cys386 (FIG. 13A). This subpocket accommodates the C1′-gem-dimethyl group introduced in manysynthetic cannabinoids, accounting for a notable enhancement in potencyand efficacy observed for these homologues.⁴⁷⁻⁵⁰ Althoughstructure-activity relationship (SAR) studies with classicalcannabinoids have shown that the C3 alkyl chain lengths modulate theligand affinity at CB1 receptor, (−)-trans-Δ⁹-THCB was 3-times moreactive than (−)-trans-Δ⁹-THC. Interestingly, from docking calculationthe butyl chain of THCB does not extend within the tunnel, such as forTHC. In contrast, it accommodates within the sub-pocket, maximizing thehydrophobic interaction with Phe170, Phe200, and Leu387 (FIG. 13B) andaccounting for the higher affinity against CB1 than Δ⁹-THC and Δ⁹-THCV.

2.2.5 Semi-Quantification of CBDB and Δ⁹-THCB in the FM2 Variety

The concentration of total CBD and total THC in the FM2 variety measuredby gas chromatography coupled to flame ionization detector (GC-FID)provided in the certificate of analysis by the MilitaryChemical-Pharmaceutical Institute was 59 mg/g and 42 mg/g respectively.In order to provide an approximate concentration of their butylhomologues, a semi-quantitative analysis was carried out by performingan external calibration for each analyte, including CBD, Δ⁹-THC, CBDBand Δ⁹-THCB. The FM2 ethanol extract was injected into theHPLC-Q-Exactive system and the concentration calculated for CBDB andΔ⁹-THCB was 0.5 mg/g and 0.4 mg/g (w/w) respectively. The valuesobtained for CBD and Δ⁹-THC (56 and 39 mg/g respectively) were inaccordance with those provided by the Institute of Florence.

2.2.6 Effect of the New Compounds on the Formalin Test in Mice

Nociceptive response to subcutaneous formalin induced an early, shortlasting first phase (0-7 min) followed by a quiescent period and then asecond, prolonged phase (15-60 min) of tonic hyperalgesia. Systemicadministration of Δ⁹-THCB (3 mg/kg, i.p.) (F_((3,12))=2.09 P=0.15)reduced both first and second phase of formalin test (FIG. 14A).Pre-treatment with both AM251 (F_((3,12))=6.23 P=0.01) and AM630(F_((3,12))=4.14 P=0.03) prevented the analgesic effects induced byΔ⁹-THCB (FIG. 14B-C). These data suggest that the pharmacological effectof Δ⁹-THCB is likely to be mediated by the cannabinoid system.Interestingly, the effect on the first phase of the formalin test, couldalso suggest us that other mechanisms at the basis of Δ⁹-THCBpharmacological action might be involved. The possible involvement ofother receptors such as Transient Receptors Potential channels family(TRPs) might also explain, at least in part, biphasic effect atdifferent doses.⁵¹ Indeed, Δ⁹-THCB is effective at 3 mg/Kg, whereas itspharmacological effect in preventing formalin-induced nocifensivebehavior is similar or even reduced. Further studies may be investigatethe potential effect of this compound on other type of chronic painincluding neuropathic pain.

2.2.7 In Vivo Determination of the Cannabimimetic Profile of Δ⁹-THCB

The cannabinoid activity of Δ⁹-THCB was evaluated by the tetrad ofbehavioral tests on mice. The tetrad includes the assessment ofspontaneous activity, immobility index (catalepsy), analgesia andchanges in rectal temperature. Decrease of locomotor activity,catalepsy, analgesia and hypothermia are well-known signs ofphysiological manifestations of cannabinoid or cannabimimetic activity.After intraperitoneal (i.p.) administration (FIG. 15A), Δ⁹-THCB at 10and 20 mg/kg did not show any alteration on the spontaneous activity ofmice in the open field, (FIG. 15B) (Naîve/veh: 3283 cm±390.5, 10 mg/kg:3220 cm±212.4, 20 mg/kg: 3591 cm±597.9, P=0.7061). Moreover, Δ⁹-THCBadministration induced a significant increase, only at 20 mg/kg, in thelatency for moving from the catalepsy bar (FIG. 15C) (Naïve/veh: 3.50sec+0.65, 10 mg/kg: 3.0 sec+0.82, 20 mg/kg: 13.0 sec+0.91, P=0.0074). Inthe hot plate test (FIG. 15F), Δ⁹-THCB administration (only at 20 mg/kg)induced a significant antinociceptive effect as compared to the vehicletreated mice (Naïve/veh: 10.15 sec+0.70, 10 mg/kg: 7.94 sec+0.06, 20mg/kg: 17.50 sec±2.53, p=0.0006) (FIG. 15D). On the other hand, Δ⁹-THCBadministration did not induce significant changes in the rectaltemperature (Naïve/veh: 0.53° C.±0.21, 10 mg/kg: −0.28° C.±0.10, 20mg/kg: 0.40° C.±0.35, p=0.0379) (FIG. 15E). These results suggest apartial interaction of Δ⁹-THCB with CB1 receptor, at least in naïveconditions. On the other hand, the formalin test indicates that thecapability of this new cannabinoid in modulating the CB receptors isincreased in a pathological state. Further studies are ongoing toelucidate the actual pharmacological mechanism of action of Δ⁹-THCB andCBDB.

2.2.8 Concluding Remarks

The butyl homologues of CBD (CBDB) and Δ⁹-THC (Δ⁹-THCB) were isolatedfrom a medicinal Cannabis variety. For the first time their absoluteconfiguration was assigned and their chemical and spectroscopicproperties were characterized and compared to those of authenticstandards obtained via a stereoselective synthesis. The results obtainedwith the biological tests indicated that the binding affinity of Δ⁹-THCBfor hCB1 is similar to that of Δ⁹-THC and higher than that of Δ⁹-THCV.Docking studies suggested that the fitting of Δ⁹-THCB into hCB1 receptorpocket is different from that of Δ⁹-THC, thus justifying the outcome ofthe binding experiments. Moreover, Δ⁹-THCB showed analgesic effects inthe formalin test in mice, although more in-depth investigation mayprovide additional understanding its potential effects in differenttypes of chronic pain. The results of the tetrad test indicated thatΔ⁹-THCB should be a partial agonist for CB₁ receptor although furtherstudies may be helpful to completely disclose its pharmacologicalmechanism in vivo. For the pharmacological research, Δ⁹-THCB and CBDBmight represent two new phytocannabinoids to focus on in the near futurein order to unveil the disconcerting plethora of medicinal properties ofCannabis.

2.3 Experimental Section 2.3.1 General Experimental Procedures

Ethanol 96% (analytical grade), acetonitrile, water and formic acid(LC-MS grade) were purchased from Carlo Erba (Milan, Italy).Δ⁹-Tetrahydrocannabivarin (Δ⁹-THCV), Δ⁹-tetrahydrocannabinol (Δ⁹-THC)and cannabidiol (CBD) were purchased as Cerilliant certified analyticalstandards (Sigma-Aldrich, Milan, Italy).(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol and5-butylbenzene-1,3-diol were purchased from Combi-blocks (San Diego,Calif., USA) and GreenPharma (Foligno, Italy), respectively. Chemicalsand solvents for the synthesis were reagent grade and used withoutfurther purification. Reactions were monitored by thin-layerchromatography on silica gel plates (60F-254, E. Merck) and visualizedwith UV light, or alkaline KMnO₄ aqueous solution. NMR spectra wererecorded on a Bruker 400 spectrometer with ¹H at 400.134 MHz and ¹³C at100.62 MHz or with a Bruker 600 spectrometer with ¹H at 600.130 MHz and¹³C at 150.902 MHz. Proton chemical shifts were referenced to thesolvent residual peaks (CDCl₃ 6=7.26 ppm). Chemical shifts are reportedin parts per million (ppm, 6 units). Coupling constants are reported inhertz (Hz). Splitting patterns are designed as s, singlet; d, doublet;t, triplet; q quartet; dd, double doublet; m, multiplet; b, broad. ¹HNMR were acquired with a spectral width of 8278 Hz, a relaxation delayof 1 s, and 32 number of transient. ¹³C NMR were acquired with aspectral width of 23.9 kHz, a relaxation delay of 1 s, and 1024 and 6144number of transient for synthetic Δ⁹-THCB and extracted Δ⁹-THCB,respectively. Carbon chemical shifts were reported in parts per million(ppm, 6 units) and referenced to the solvent residual peaks (CDCl₃6=77.20 ppm). The COSY were recorded as a 2048×256 matrix with 2transients per t1 increment and processed as a 2048×1024 matrix. TheHSQC spectra were collected as a 2048×256 matrix with 4 transients pert1 increment and processed as a 2048×1024 matrix, and the one-bondheteronuclear coupling value was set to 145 Hz. The HMBC spectra werecollected as a 4096×256 matrix with 16 transients per t1 increment andprocessed as a 4096×1024 matrix, and the long-range coupling value wasset to 8 Hz.

(6aR,10aR)-3-butyl-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol,(−)-trans-Δ⁸-tetrahydrocannabidibutol (Δ⁸-THCB)

To a solution of 5-butylbenzene-1,3-diol (332 mg, 2.0 mmol, 1 eq.) andp-toluenesulfonic acid (40 mg, 0.2 mmol, 0.1 eq.) in dry dichloromethane(CH₂Cl₂) (10 mL) at room temperature, under argon atmosphere, a solutionof (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol (314 mg, 2.0 mmol,1 eq.) in 10 mL of dry CH₂Cl₂ was added dropwise. The mixture wasstirred at room temperature for 2 days and then quenched with asaturated solution of NaHCO₃ (10 mL). The resulting mixture wasextracted in diethyl ether (2×10 mL). The combined organic phases werecollected, washed with brine, dried over anhydrous Na₂SO₄ andconcentrated. The crude was purified over silica gel (crude:silica ratio1:200, eluent: cyclohexane:CH₂Cl₂ 7:3) to give 260 mg of a reddish oil(43% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.28 (1H, d, J=1.4 Hz), 6.10 (1H,d, J=1.5 Hz), 5.49-5.34 (1H, m), 4.62 (1H, s), 3.19 (1H, dd, J=4.5, 15.8Hz), 2.70 (1H, td, J=4.7, 10.8 Hz), 2.45 (2H, td, J=2.5, 7.5 Hz),2.21-2.07 (1H, m), 1.90-1.75 (3H, m), 1.73-1.68 (3H, m), 1.63-1.50 (2H,m), 1.39-1.29 (5H, m), 1.11 (3H, s), 0.91 (3H, t, J=7.3 Hz). HRESIMS m/z301.2165 [M+H]+(calcd for C₂₀H₂₉O₂ ⁺, 301.2162).

(6aR,10aR)-3-butyl-9-chloro-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol(HCl-THCB)

To a solution of Δ⁸-THCB (260 mg, 0.87 mmol, 1 eq.) in dry CH₂Cl₂ (10mL) at room temperature and under nitrogen atmosphere, ZnCl₂ iN in Et₂O(430 μL, 0.43 mmol, 0.5 eq.) was added. The mixture was stirred in thesame condition for 30 minutes and then cooled at 0° C. A large excess ofHCl 4N in dioxane (1 mL) was added, the temperature spontaneously raisedat room temperature and left reacted for 24 h. The solvent wasevaporated and the residue re-solubilized in diethyl ether. The organiclayer was washed with a saturated solution of NaHCO₃, brine, dried overanhydrous Na₂SO₄ and concentrated to give 293 mg of a yellowish oil(quant. yield). ¹H NMR (400 MHz, CDCl₃) δ 6.25 (1H, s), 6.08 (1H, s),4.63 (1H, s), 3.71 (3H, s), 3.44 (1H, dt, J=2.8, 14.2 Hz), 3.05 (1H, td,J=2.9, 11.3 Hz), 2.44 (2H, dd, J=6.4, 8.9 Hz), 2.17 (1H, dt, J=2.6, 10.7Hz), 1.79-1.28 (10H, m), 1.13 (3H, s), 0.90 (3H, t, J=7.3 Hz). ¹³C NMR(101 MHz, CDCl₃) δ 13.95, 19.15, 22.36, 24.24, 27.70, 31.37, 33.03,34.23, 35.08, 42.08, 44.88, 48.76, 67.10, 72.63, 107.67, 108.91, 110.14,142.83, 154.45, 155.09. HRESIMS m/z 339.1900 [M+H]+(calcd for C₂₀H₃₀³⁷[Cl]O₂+, 339.1899), m/z 337.1932 [M−H]⁻ (calcd for C₂₀H₃₀ ³⁵[Cl]O₂,337.1929).

(6aR,10aR)-3-butyl-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol,(−)-trans-Δ⁹-THCB

To a solution of 1.75 N potassium t-amylate in toluene (1.23 mL, 2.15mmol, 2.5 eq.), in dry toluene (10 mL) at −15° C. and under argonatmosphere, a solution of HCl-THCB (290 mg, 0.86 mmol, 1 eq.) in drytoluene (10 mL) was added. The mixture was stirred in the same conditionfor 30 minutes and then at room temperature for 1 h. The mixture wasdiluted with diethyl ether and quenched with a 1% solution of ascorbicacid. The organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated to give 235 mg of a greenish oil (91% yield). 10mg of (−)-trans-Δ⁹-THCB were further purified by semipreparative HPLC toprepare a pure analytic standard (purity >99.9%). [α]_(D) ²⁰=−1430 (c2.3, ACN). ¹H NMR (400 MHz, CDCl₃) δ 6.30 (1H, dt, J=1.7, 3.6 Hz), 6.27(1H, d, J=1.7 Hz), 6.14 (1H, d, J=1.7 Hz), 4.84 (1H, bs), 3.22 (1H, dt,J=2.8, 10.8 Hz), 2.47 (2H, td, J=1.7, 7.3 Hz), 2.26-2.16 (2H, m),2.02-1.90 (1H, m), 1.68 (3H, t, J=1.8 Hz), 1.58-1.49 (2H, m), 1.45-1.30(7H, m), 1.11 (3H, s), 0.92 (3H, t, J=7.3 Hz). ¹³C NMR (101 MHz, CDCl₃)δ 154.76, 154.17, 142.75, 134.38, 123.72, 110.09, 109.03, 107.54, 77.20,45.81, 35.17, 33.58, 33.11, 31.17, 27.57, 25.02, 23.36, 22.34, 19.27,13.95. HRESIMS m/z 301.2157 [M+H]⁺ (calcd for C₂₀H₂₉O₂ ⁺, 301.2162), m/z299.2016 [M−H]⁻ (calcd for C₂₀H₂₇O₂, 299.2017).

Plant Material. FM2 Cannabis inflorescence (batch n. 6A32/1) wasobtained by the Military Chemical Pharmaceutical Institute (Firenze,Italy) with the authorization of the Italian Minister of Health (prot.n. SP/062). The inflorescence (5 g) was finely ground and divided intotwo batches: a 500 mg batch was extracted with 50 mL of ethanol 96% eachaccording to the procedure indicated by the monograph of Cannabis Flosof the German Pharmacopoeia⁵². The remainder (4.5 g) was subjected todecarboxylation in oven at 120° C. for 2 h. A 500 mg aliquot of thisbatch was extracted as the previous one and both were analyzed byUHPLC-HESI-Orbitrap after proper dilution (×10). The remaining 4 g weredissolved into 40 mL of ethanol 96% and used for isolation of Δ⁹-THCB bysemi-preparative liquid chromatography.

Isolation of Natural CBDB and Δ⁹-THCB. A sample of FM2 inflorescence (4g) was dissolved in ethanol 96% (40 mL) and 0.5 mL aliquots of thesolution were injected in a semi-preparative LC system (Octave 10 SembaBioscience, Madison, USA). The chromatographic conditions used arereported in the paper by Citti et al.²⁷ The column employed was a LunaC18 with a fully porous silica stationary phase (Luna 5 μm C18(2) 100 Å,250×10 mm) (Phenomenex, Bologna, Italy) and a mixture ofacetronitrile:0.1% aqueous formic acid 70:30 (v/v) was used as mobilephase at a flow rate of 5 mL/min. CBDB and Δ⁹-THCB (retention time 18.7min and 34.0 min respectively) were isolated as reported in our previouswork.²⁷ The fractions containing CBDB and Δ⁹-THCB were analyzed byUHPLC-HESI-Orbitrap. The fractions containing exclusively either one orthe other cannabinoid were separately combined and dried on therotavapor at 70° C. An amount of about 1 mg of CBDB and about 1 mg ofΔ⁹-THCB were obtained, both as reddish oils.

UHPLC-HESI-Orbitrap Metabolomic Analysis. The analyses of FM2 plantmaterial were performed on a Thermo Fisher Scientific Ultimate 3000equipped with a vacuum degasser, a binary pump, a thermostatedautosampler, a thermostated column compartment and a Q-Exactive Orbitrapmass spectrometer with a heated electrospray ionization source(UHPLC-HESI-Orbitrap). The direct infusion of the single analytes (1μg/mL) at 0.1 mL/min was employed to optimize the parameters of the massspectrometer. The HESI parameters were set as follows: capillarytemperature, 320° C.; vaporizer temperature, 280° C.; electrosprayvoltage, 4.2 kV (positive mode) and 3.8 kV (negative mode); sheath gas,55 arbitrary units; auxiliary gas, 30 arbitrary units; S lens RF level,45. The Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose,Calif., USA) was used to control online analyses. The analyses wereacquired in full scan data-dependent acquisition (FS-dd-MS²) in positiveand negative mode at a resolving power of 70,000 FWHM at m/z 200. Theother mass analyzer parameters were: scan range, m/z 250-400; AGC, 3e6;injection time, 100 ms; isolation window for the filtration of theprecursor ions, m/z 2. Fragmentation of precursors was performed at 30as normalized collision energy (NCE) by injecting a standard mixture ofthe analytes at a concentration of 1 μg/L. Detection was based oncalculated [M+H]⁺ and [M−H]⁻ molecular ions with an accuracy of 2 ppm,retention time and MS/MS spectrum match with pure analytical standards.

The chromatographic separation was performed on a Poroshell 120 SB-C18(3.0×100 mm, 2.7 μm, Agilent, Milan, Italy) eluting 0.1% aqueous formicacid (A) and acetonitrile (B) as mobile phase. A linear gradient from 5%to 95% B was set over 20 min, followed by an isocratic elution at 95% Bfor 5 min, and re-equilibration to the initial conditions (5% B) forfurther 5 min. The flow rate was maintained constant at 0.5 mL/min andthe injection volume was 5 μL.

In order to provide a semi-quantitative analysis of the butyl homologuesof CBD and Δ⁹-THC, we performed a calibration curve with externalstandard. A stock solution of CBDB and Δ⁹-THCB (1 mg/mL) were properlydiluted to obtain 5 calibration standards with the final concentrationsof 0.50, 1.50, 3.75, 7.50 and 11.25 μg/mL for both analytes. Thelinearity was assessed by the coefficient of determination, which wasgreater than 0.998 for both analytes.

Binding at CB1 and CB2 Receptors. The binding affinity of(−)-trans-Δ⁹-THCB against human CB1 and CB2 receptors was determined ina radioligand binding assay performed by Eurofins Discovery. Thecompound was tested at ten concentrations, ranging from 1 nM to 30 μM,in duplicate. The compound binding was calculated as a % inhibition ofthe binding of a radioactively labeled ligand specific for each target.[3H]CP55940 (at 2 nM, K_(d)=2.4 nM)⁵³ and [³H]WIN 55212-2 (at 0.8 nM,K_(d)=1.5 nM)⁵⁴ were used as specific radioligand for hCB1 and hCB2,respectively. The results were expressed as a percent inhibition ofcontrol specific binding obtained in the presence of the testedcompounds using eq. 1.

$\begin{matrix}{{\%\mspace{14mu}{in}} = {100 - \left( {\frac{{measured}\mspace{14mu}{specific}\mspace{14mu}{binding}}{{control}\mspace{14mu}{specific}\mspace{14mu}{binding}}*100} \right)}} & {{eq}.\mspace{11mu} 1}\end{matrix}$

The IC₅₀ values (concentration causing a half-maximal inhibition ofcontrol specific binding) were determined by non-linear regressionanalysis of the competition curves generated with mean replicate values(eq. 2

$\begin{matrix}{Y = {D + \left\lbrack \frac{A - D}{1 + {\left( \frac{C}{C50} \right){nH}}} \right\rbrack}} & {{eq}.\mspace{11mu} 2}\end{matrix}$

where Y is the specific binding, A is the left asymptote of the curve, Dis the right asymptote f the curve, C is the compound concentration, C₅₀is the IC₅₀ value and nH is the slope factor. This analysis wasperformed using software developed at Cerep (Hill software) andvalidated by comparison with data generated by the commercial softwareSigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.). The inhibitionconstants (K_(i)) were calculated using the Cheng Prusoff equation:

$\begin{matrix}{{Ki} = \frac{I\; C_{50}}{\left( {1 + \frac{L}{K_{D}}} \right)}} & {{eq}.\mspace{11mu} 3}\end{matrix}$

where L is the concentration of radioligand in the assay, and K_(D) isthe affinity of the radioligand for the receptor.

CP 55940 (CB1 IC₅₀=1.7 nM, CB1 K_(i)=0.93 nM) and WIN 55212-2 (CB2IC₅₀=2.7 nM, CB2 K_(i)=1.7 nM) were used as reference compounds againsthCB1 and hCB2, respectively and the results are in accordance with thevalues reported in literature.^(53, 54)

Docking calculation. The crystal structure for the active conformationof CB1 (PDB ID: 5XRA) was used as reference protein for dockingcalculation. The protein was prepared using the protein preparationwizard module of the Schrodinger suite⁵⁶. The protonation and tautomericstates of the residues were adjusted at pH 7.0^(57, 58). Water moleculeswere removed, and the hydrogens position was minimized with theOPLS_2005 force field. The chemical structures ofΔ⁹-tetrahydrocannabinol (Δ⁹-THC), Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV),and Δ⁹-tetrahydrocannabidibutol (Δ⁹-THCB), were drawn using ChemDraw12.0 and prepared using the LigPrep module of Schrödinger suite with theOPLS_2005 force field⁵⁹. The three ligands were docked into theorthosteric binding site of the active conformation of the CB1 structureby using the Glide module of Schrödinger suite⁶⁰.

Formalin test in mice. Male CD-1 mice 6-8 weeks (Envigo, Italy), werehoused under controlled conditions (12 h light/12 h dark cycle;temperature 20-22 C; humidity 55-60%) with chow and tap water availablead libitum. All surgeries and experimental procedures were approved bythe Animal Ethics Committee of University of Campania “L. Vanvitelli”(Naples). Animal care was in compliance with Italian (D.L. 116/92) andEuropean Commission (O.J. of E.C. L358/1 18/12/86) regulations on theprotection of laboratory animals. All efforts were made to minimizeanimal suffering and to reduce the number of animals used. All theexperiments were performed in a randomized manner by the same operatorblind to pharmacological treatments. Mice weighing 28-32 g were usedafter 1 week-acclimation period and received formalin (1.25% in saline,30 μL) in the dorsal surface of one side of the hind-paw. Each mouse,randomly assigned to one of the experimental groups (n=5), was placed ina plexiglass cage and allowed to move freely for 15-20 min. A mirror wasplaced at a 45° angle under the cage to allow full view of thehind-paws. Lifting, favoring, licking, shaking and flinching of theinjected paw were recorded as nocifensive behavior.⁶¹ The total time ofthe nociceptive response was measured every 5 min for 60 minutes andexpressed in min (mean±SEM). Mice received vehicle (0.5% DMSO in saline)or different doses of Δ⁹-THCB (2, 3 and 5 mg/kg, i.p.) 20 min beforeformalin injection alone or in combination with AM251 (0.5 mg/kg, i.p.)or AM630 (1 mg/kg, i.p.). The antagonists were administered 10 minbefore drugs injection.

Tetrad test. Male C57BL6/J mice 6-8 weeks (Envigo, Italy) were housedunder controlled conditions (12 h light/12 h dark cycle; temperature20-22 C; humidity 55-60%) with chow and tap water available ad libitum.All surgeries and experimental procedures were approved by the AnimalEthics Committee of University of Campania “L. Vanvitelli,” Naples.Animal care was in compliance with Italian (D.L. 116/92) and EuropeanCommission (O.J. of E.C. L358/1 18/12/86) regulations on the protectionof laboratory animals. All efforts were made to minimize animalsuffering and to reduce the number of animals used. All the experimentswere performed in a randomized manner by the same operator blind topharmacological treatments.

Mice (n=4) were treated with Δ⁹-THCB (10 and 20 mg/kg) or vehicle (0.9%saline and 0.05% DMSO) by intraperitoneal (i.p.) administration. Micewere evaluated for hypomotility (open field test), hypothermia (bodytemperature test), antinociceptive (hot plate test), and catalepticeffects (bar test), using the tetrad tests as described in the protocolby Metna-Laurent M. et al.⁶² Statistical analysis was performed usingthe Kruskall-Wallis test and Dunn's post hoc tests.

Body temperature test. A probe was gently inserted for 1 cm into themouse rectum after the animal had been immobilized. The probe was washedand cleaned with 70% ethanol and the dried with a paper towel. The bodytemperature was measured after stabilization of the value in ° C. inbasal conditions and at 50 minutes after drug or vehicle administration.

Open field test. The open field test (OFT) was performed 30 min afterdrug or vehicle injection. The apparatus was cleaned before each testusing a 70% EtOH solution. After randomly assigning naïve mice to atreatment group, the operator started to record animal behaviors, whichwere stored and analyzed using an automated behavioral tracking system(Smart v3.0, Panlab Harvard Apparatus). Mice were placed in an OFT arena(l×w×h: 44 cm×44 cm×30 cm), and ambulatory activity (total distancetravelled in cm), was recorded for 15 min and analyzed.

Bar test. The bar used was a 40 cm long and 0.4 cm wide in diameterglass rod, which was horizontally elevated by 5 cm above the surface.Both forelimbs of the mouse were positioned on the bar and its hind legson the floor of the cage, ensuring that the animal was not lying down onthe floor. The chronometer was stopped when the mouse descended from thebar (i.e., when the two forepaws touched the floor) or after the 10 mincut-off time. Catalepsy was measured as the time duration each mouseheld the bar by both his forelimbs (latency for moving in seconds).

Hot plate test. Each mouse was placed on a hot plate (Ugo Basile), whichwas kept at a constant temperature of 52° C. The nociceptive response(NR) was recorded as characteristic actions of the mouse like licking ofthe hind paws, as well as jumping. The latency to the NR was measured insec 85 minutes after drug or vehicle administration. A 30 or 60 seccut-off time was used in order to prevent tissue damage.

Statistics for in vivo experiments. Data are represented asmeans±standard error of the mean (S.E.M). Statistical analysis of thedata was performed by analysis of variance two-way ANOVA followed byBonferroni post hoc or Dunnett's multiple comparison test. Allstatistical analyses were performed by using GraphPAD software (SanDiego, Calif.). Differences were considered significant at p<0.05.

2.4 Associated Content

Exhibit B—Supporting Information and FIG. 16. Spectroscopic data ofsynthetic and extracted Δ⁹-THCB: original NMR and circular dichroism(CD) spectra for the new compound.

2.5 References

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Example 3 A Novel Phytocannabinoid Isolated from Cannabis sativa L. Withan In Vivo Cannabimimetic Activity Higher than Δ⁹-tetrahydrocannabinol:Δ⁹-Tetrahydrocannabiphorol Abstract

(−)-Trans-Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the main compoundresponsible for the intoxicant activity of Cannabis sativa L. The lengthof the side alkyl chain influences the biological activity of thiscannabinoid. In particular, synthetic analogues of Δ⁹-THC with a longerside chain have shown cannabimimetic properties far higher than Δ⁹-THCitself. In the attempt to define the phytocannabinoids profile thatcharacterizes a medicinal Cannabis variety, a new phytocannabinoid withthe same structure of Δ⁹-THC but with a seven-term alkyl side chain wasidentified. The natural compound was isolated and fully characterizedand its stereochemical configuration was assigned by match with the samecompound obtained by a stereoselective synthesis. This newphytocannabinoid has been called (−)-trans-Δ⁹-tetrahydrocannaphorol(Δ⁹-THCP). Along with Δ⁹-THCP, the corresponding cannabidiol (CBD)analog with seven-term side alkyl chain was also isolated and identifiedby match with its synthetic counterpart. The binding activity of Δ⁹-THCPagainst human CB₁ receptor in vitro (K_(i)=1.2 nM) resulted similar tothat of CP55940 (K_(i)=0.9 nM), a potent full CB₁ agonist. In thecannabinoid tetrad pharmacological test, Δ⁹-THCP induced hypomotility,analgesia, catalepsy and decreased rectal temperature indicating aTHC-like cannabimimetic activity. The presence of this newphytocannabinoid could account for the high efficacy of some extremelypotent Cannabis varieties.

Introduction

Cannabis sativa has always been a controversial plant due to itspositive and negative implications, a lifesaver for several pathologiesincluding glaucoma¹ and epilepsy², an invaluable source of nutrients³,an environmentally friendly raw material for manufacturing⁴ andtextiles⁵, but it is also the most widely spread illicit drug in theworld, especially among young adults⁶.

A peculiarity is its ability to produce a class of organic moleculescalled phytocannabinoids, which derive from an enzymatic reactionbetween a resorcinol and an isoprenoid group. The modularity of thesetwo parts is the key for the extreme variability of the resultingproduct that has led to almost 150 different known phytocannabinoids⁷.The precursors for the most commonly naturally occurringphytocannabinoids are olivetolic acid and geranyl pyrophosphate, whichtake part to a condensation reaction leading to the formation ofcannabigerolic acid (CBGA). CBGA can be then converted into eithertetrahydrocannabinolic acid (THCA) or cannabinolic acid (CBDA) orcannabichromenic acid (CBCA) by the action of a specific cyclaseenzyme⁷. All phytocannabinoids are biosynthesized in the carboxylatedform, which can be converted into the corresponding decarboxylated (orneutral) form by heat⁸. The best known neutral cannabinoids areundoubtedly Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD), theformer being responsible for the intoxicant properties of Cannabisplant, and the latter being active as antioxidant, anti-inflammatory,anti-convulsant, but also as antagonist of THC negative effects⁹.

All these cannabinoids are characterized by the presence of an alkylside chain on the resorcinyl moiety made of five carbon atoms. However,other phytocannabinoids with a different number of carbon atoms on theside chain are known and they have been called varinoids (with threecarbon atoms), such as cannabidivarin (CBDV) andΔ⁹-tetrahydrocannabivarin (Δ⁹-THCV), and orcinoids (with one carbonatom), such as cannabidiorcol (CBD-C₁) and tetrahydrocannabiorcol(THC-C₁)⁷. Both series are biosynthesized in the plant as the specificketide synthases have been identified¹⁰.

Our research group has recently reported the presence of a butylphytocannabinoid series with a four-term alkyl chain, in particularcannabidibutol (CBDB) and Δ⁹-tetrahydrocannabutol (Δ⁹-THCB), in CBDsamples derived from hemp and in a medicinal Cannabis variety^(11,12).Since no evidence has been provided for the presence of plant enzymesresponsible for the biosynthesis of these butyl phytocannabinoids, ithas been suggested that they might derive from microbial ω-oxidation anddecarboxylation of their corresponding five-term homologues¹³.

The length of the alkyl side chain has indeed proved to be the keyparameter, the pharmacophore, for the biological activity exerted byΔ⁹-THC on the human cannabinoid receptor CB₁ as evidenced bystructure-activity relationship studies collected by Bow and Rimondi¹⁴.In particular, a minimum of three carbons is necessary to bind thereceptor, then the highest activity has been registered with aneight-carbon side chain to finally decrease with a higher number ofcarbon atoms¹⁴. Δ⁸-THC homologues with more than five carbon atoms onthe side chain have been synthetically produced and tested in order tohave molecules several times more potent than Δ⁹-THC¹⁵.

To the best of our knowledge, a phytocannabinoid with a linear alkylside chain containing more than five carbon atoms has never beenreported as naturally occurring. However, our research group disclosedfor the first time the presence of seven-term homologues of CBD andΔ⁹-THC in a medicinal Cannabis variety, the Italian FM2, provided by theMilitary Chemical Pharmaceutical Institute in Florence. The two newphytocannabinoids were isolated and fully characterized and theirabsolute configuration was determined by a stereoselective synthesis.According to the International Non-proprietary Name (INN), we suggestedfor these CBD and THC analogues the name “cannabidiphorol” (CBDP) and“tetrahydrocannabiphorol” (THCP), respectively. The suffix “-phorol”comes from “sphaerophorol”, common name for 5-heptyl-benzen-1,3-diol,which constitutes the resorcinyl moiety of these two newphytocannabinoids. In particular, the seven-term Δ⁹-THC homologue showedsurprisingly higher binding affinity to CB₁ in vitro and highercannabimimetic activity in vivo in the tetrad test compared to Δ⁹-THC.

3.1 Results 3.1.1 Identification of Cannabidiphorol (CBDP) andΔ⁹-tetrahydrocannabiphorol (THCP) by Liquid Chromatography Coupled toHigh-Resolution Mass Spectrometry (LC-HRMS)

The FM2 ethanolic extract was analyzed by an analytical method recentlydeveloped for the cannabinoid profiling of this medicinal Cannabisvariety^(12,16). As the native extract contains mainly the carboxylatedforms of phytocannabinoids as a consequence of a cold extraction¹⁷, partof the plant material was heated to achieve decarboxylation where thepredominant forms are neutral phytocannabinoids. The advanced analyticalplatform of ultra-high performance liquid chromatography coupled to highresolution Orbitrap mass spectrometry was employed to analyze the FM2extracts and study the fragmentation spectra of the analytes underinvestigation. The precursor ions of the neutral derivativescannabidiphorol (CBDP) and Δ⁹-tetrahydrocannabiphorol (Δ⁹-THCP),341.2486 for the [M−H]⁻ and 343.2632 for the [M+H]⁺, showed an elutiontime of 19.4 for CBDP and 21.3 for Δ⁹-THCP (FIG. 17a ). Theiridentification was determined by the injection of a mixture (5 ng/mL) ofthe two chemically synthesized CBDP and Δ⁹-THCP (FIG. 17b ) as it willbe described later. As for their carboxylated counterpart, the precursorions of the neutral forms CBDP and Δ⁹-THCP break in the same way in ESI+mode, but they show a different fragmentation pattern in ESI− mode.Whilst Δ⁹-THCP shows only the precursor ion [M−H]⁻ (FIG. 17d ), CBDPmolecule generates the fragments at m/z 273.1858 corresponding to aretro Diels-Alder reaction, and 207.1381 corresponding to the resorcinylmoiety after the break of the bond with the terpenoid group (FIG. 17c ).It is noteworthy that for both molecules, CBDP and Δ⁹-THCP, eachfragment in both ionization modes differ exactly by an ethylene unit(CH₂)₂ from the corresponding five-termed homologues CBD and THC.Moreover, the longer elution time corroborate the hypothesis of theseven-termed phytocannabinoids considering the higher lipophilicity ofthe latter.

3.1.2 Isolation and Characterization of CBDP and Δ⁹-THCP

In order to selectively obtain a cannabinoid-rich fraction of FM2,n-hexane was used to extract the raw material instead of ethanol, whichcarries other contaminants such as flavonoids and chlorophylls alongwith cannabinoids¹⁸. An additional dewaxing step at −20° C. for 48 h andremoval of the precipitated wax resulted in a pure cannabinoids extract.Semi-preparative liquid chromatography with a Cis stationary phaseallowed for the separation of 80 fractions, which were analyzed byLC-HRMS with the previously described method. In this way, the fractionscontaining predominantly CBDPA and THCPA were separately subject toheating at 120° C. for 2 h in order to obtain their correspondingneutral counterparts CBDP and Δ⁹-THCP as clear oils with a >95% purity.The material obtained was sufficient for a full characterization by ¹Hand ¹³C NMR, circular dichroism (CD) and UV absorption.

3.1.3 Stereoselective Synthesis of CBDP and THCP

(−)-trans-Cannabidiphorol ((−)-trans-CBDP) and(−)-trans-Δ⁹-tetrahydrocannabiphorol ((−)-trans-Δ⁹-THCP) werestereoselectively synthesized as previously reported for the synthesisof (−)-trans-CBDB and (−)-trans-Δ⁹-THCB homologues^(11,16). Accordingly,(−)-trans-CBDP was prepared by condensation of 5-heptylbenzene-1,3-diol(1) with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using pTSAas catalyst, for 90 min. Longer reaction time did not improve the yieldof (−)-trans-CBDP because cyclization of (−)-trans-CBDP to(−)-trans-Δ⁹-THCP and then to (−)-trans-Δ⁸-THCP occurs.5-heptylbenzene-1,3-diol (1) was synthesized first as reported in theExhibit C—Supporting Information (Supplementary FIG. SI-1). Theconversion of (−)-trans-CBDP to (−)-trans-Δ⁹-THCP using diverse Lewis'acids, as already reported in the literature for the synthesis of thehomologue Δ⁹-THC¹⁹⁻²¹, led to a complex mixture of isomers which resultsin an arduous and low-yield isolation of (−)-trans-Δ⁹-THCP by standardchromatographic techniques. Therefore, for the synthesis of(−)-trans-Δ⁹-THCP, its regioisomer (−)-trans-Δ⁸-THCP was synthesizedfirst by condensation of 5-heptylbenzene-1,3-diol with(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, as described above,but the reaction was left stirring for 48 hours. Alternatively,(−)-trans-CBDP could be also quantitatively converted to(−)-trans-Δ⁸-THCP in the same conditions. Hydrochlorination of the Δ⁸double bond of (−)-trans-Δ⁸-THCP, using ZnCl₂ as catalyst, allowed toobtain (−)-trans-HCl-THCP, which was successively converted to(−)-trans-Δ⁹-THCP in 87% yield by selective elimination on 2 position ofthe terpene moiety using potassium t-amylate as base (FIG. 18a ).

The chemical identification of synthetic (−)-trans-CBDP and(−)-trans-Δ⁹-THCP, and its ¹H and ¹³C assignments were achieved by NMRspectroscopy (Supplementary Table SI-1,2 and Supplementary FIG. SI-2,3in Exhibit C). Since (−)-trans-CBDP and (−)-trans-Δ⁹-THCP differ fromthe respective homologues (CBD, CBDB, CBDV, Δ⁹-THC, Δ⁹-THCB and Δ⁹-THCV)solely for the length of the alkyl chain on the resorcinyl moiety, nosignificative differences in the proton chemical shifts of the terpeneand aromatic moieties were observed for CBD and Δ⁹-THC homologues. Theperfect match in the chemical shift of the terpene and aromatic amongthe synthesized (−)-trans-CBDP and (−)-trans-Δ⁹-THCP and the respectivehomologues^(11,16,22), combined with the mass spectra and fragmentationpattern, allowed us to determine the chemical structures of the two newsynthetic cannabinoids. The trans configuration at the terpene moietywas determined by optical rotatory power. The new cannabinoids(−)-trans-CBDP and (−)-trans-Δ⁹-THCP showed an [α]_(D) ²⁰ of −145° and163°, respectively, in chloroform. The [α]_(D) ²⁰ values are in linewith those of the homologues^(11,23), suggesting a (1R,6R) configurationfor both CBDP and Δ⁹-THCP. A perfect superimposition between the ¹H(FIG. 18b,e ) and ¹³C NMR spectra (FIG. 18c,f ) and the circulardichroism absorption (FIG. 18d,g ) of both synthetic and extracted(−)-trans-CBDP and (−)-trans-Δ⁹-THCP was observed, determining theidentity of the two new cannabinoids identified in the FM2 Cannabisvariety.

3.1.4 Binding Affinity at Human CB1 and CB2 Receptors

The binding affinity of (−)-trans-Δ⁹-THCP against purified human CB₁ andCB₂ receptors was determined in a radioligand binding assay, using[3H]CP55940 or [3H]WIN 55212-2 as reference compounds, and dose-responsecurves were determined (FIGS. 19a and 19b ). (−)-trans-Δ⁹-THCP bindswith high affinity to both human CB₁ and CB₂ receptors with a K_(i) of1.2 and 6.2 nM, respectively. (−)-trans-Δ⁹-THCP results 33-times moreactive than (−)-trans-Δ⁹-THC (K_(i)=40 nM), 63-times more active than(−)-trans-Δ⁹-THCV (K_(i) of 75.4 nM) and 13-times more active than thenewly discovered (−)-trans-Δ⁹-THCB (K_(i) of 15 nM) against CB₁receptor^(12,14). Moreover, the new identified (−)-trans-Δ⁹-THCPresulted about 5- to 10-times more active against CB₂ receptor (K_(i) of6.2 nM), in contrast with (−)-trans-Δ⁹-THC, (−)-trans-Δ⁹-THCB and(−)-trans-Δ⁹-THCV, which instead show a comparable biding affinity witha K_(i) ranging from 36 to 63 nM (FIG. 19a )^(12,14).

The highest activity of (−)-trans-Δ⁹-THCP, compared to the shorterhomologues, was investigated by docking calculation. The X-ray structureof the active conformation of human CB₁ receptor in complex with theagonist AM11542 (PDB ID: 5XRA) was used as reference for docking sincemarked structural changes in the orthosteric ligand-binding site areobserved in comparison with the conformation of the receptor bound to anantagonist^(24,25). AM11542 is a synthetic Δ⁸ cannabinoid with highaffinity against hCB₁ receptor (K_(i)=0.11 nM) possessing a7′-bromo-1′,1′-dimethyl-heptyl aliphatic chain at C3 of the resorcinylmoiety. Due to the close chemical similarity, the predicted binding modeof (−)-trans-Δ⁹-THCP (FIG. 19c ) reflects that of AM11542 in the CB₁crystal structure (FIG. SI-6 a,b)¹⁸. (−)-trans-Δ⁹-THCP binds in theactive conformation of CB₁ in an L-shaped pose. Thetetrahydro-6H-benzo[c]chromene ring system is located within the mainhydrophobic pocket delimited by Phe174, Phe177, Phe189, Lys193, Pro269,Phe170 and Phe268. In particular, the aromatic ring of the resorcinylmoiety is involved in two edge-to-face π-π interactions with Phe170 andPhe268, whereas the phenolic hydroxyl group at C1 is engaged in a H-bondwith Ser383 (FIG. 19c ). Interestingly, the heptyl chain at C3 extendsinto a long hydrophobic tunnel formed by Leu193, Val196, Tyr275, Iso271,Leu276, Trp279, Leu359, Phe379 and Met363 (FIG. 19c,d ). Because thepredicted pose of the tricyclic tetrahydrocannabinol ring system isconserved among the four THC homologues (Supplementary FIG. SI-7 a-c inExhibit C), the length of the alkyl chain at C3 of the resorcinyl moietycould account for the different binding affinity observed among the fourcannabinoids. (−)-trans-Δ⁹-THCP (FIG. 19c ) and (−)-trans-Δ⁹-THC(Supplementary FIG. SI-7 a in Exhibit C) share the same positioning ofthe alkyl ‘tail’ within the hydrophobic channel^(12,25,26). However, thelong heptyl chain of Δ⁹-THCP is able to extend into the tunnel along itsentire length, maximizing the hydrophobic interactions with the residuesof the side channel. In contrast, the tunnel is only partially occupiedby the shorter pentyl chain of (−)-trans-Δ⁹-THC, accounting for thehigher affinity of Δ⁹-THCP (K_(i)=1.2 nM) compared to Δ⁹-THC (K_(i)=40nM). A different positioning of the ‘tail’ is instead predicted for theshorter alkyl chain homologues, Δ⁹-THCV and Δ⁹-THCB. The propyl andbutyl chain of Δ⁹-THCV and Δ⁹-THCB, respectively, are too short toeffectively extend within the hydrophobic channel. As stated in ourprevious work¹², these shorter chains accommodate within a smallhydrophobic pocket delimitated by Phe200, Leu359 and Met363(Supplementary FIG. SI-7 b,c in Exhibit C). This side pocket is locatedat the insertion between the main hydrophobic pocket and the longchannel (FIG. 19d ) and seems to accommodate small hydrophobicsubstituents (i.e. gem-dimethyl or cycloalkyl) introduced at C1′position of the side chain of several synthetic cannabinoids,rationalizing the notable enhancement in potency and affinity for thesederivatives²⁷⁻³¹.

3.1.5 In Vivo Determination of the Cannabinoid Profile of THCP

The cannabinoid activity of Δ⁹-THCP was evaluated by the tetrad ofbehavioural tests on mice. The tetrad includes the assessment ofspontaneous activity, immobility index (catalepsy), analgesia andchanges in rectal temperature. Decrease of locomotor activity,catalepsy, analgesia and hypothermia are well-known signs ofphysiological manifestations of cannabinoid activity³². Afterintraperitoneal (i.p.) administration, Δ⁹-THCP at 2.5 mg/kg markedlyreduced the spontaneous activity of mice in the open field, while at 5and 10 mg/kg it induced catalepsy on the ring with the immobility ascompared to the vehicle treated mice (FIG. 20b,c ) (0: 6888 cm±474.8, 10mg/kg: 166.8 cm±20.50, 5 mg/kg: 127.5 cm±31.32, 2.5 mg/kg: 4072cm±350.8, p=0.0009). Moreover, Δ⁹-THCP administration induced asignificant increase, at 10 and 5 mg/kg, in the latency for moving fromthe catalepsy bar (FIG. 20e ) (0: 15.20 sec±4.33, 10 mg/kg: 484.5sec±51.58, 5 mg/kg: 493.4 sec±35.68, 2.5 mg/kg: 346.1 sec+35.24,p=0.0051). In the hot plate test (FIG. 20f ), Δ⁹-THCP (10 and 5 mg/kg)induced antinociceptive effect, whereas at 2.5 mg/kg there is a trend inthe induction of antinociception, which resulted not statisticallysignificant as compared to the vehicle treated mice (0: 19.20 sec+2.65,10 mg/kg: 57.0 sec+2.0, 5 mg/kg: 54.38 sec+2.86, 2.5 mg/kg: 40.22sec+5.8, p=0.0044). Δ⁹-THCP administration induced a dose dependentsignificant decrease, only 10 mg/kg, in body temperature as compared tovehicle (0: 0.40° C.±0.25, 10 mg/kg: −7.10° C. 0.43, 5 mg/kg: −5.28°C.±0.36, 2.5 mg/kg: −4,12° C.±0.38, p=0.0009) (FIG. 20d ).

3.1.6 Semi-Quantification of CBDP and Δ⁹-THCP in the FM2 Extract

A semi-quantification method based on LC-HRMS allowed to provide anapproximate amount of the two new phytocannabinoids in the FM2 ethanolextract. Their pentyl homologues, CBD and Δ⁹-THC, showed a concentrationof 56 and 39 μg/mL respectively, in accordance with the values providedby the Military Chemical Pharmaceutical Institute (59 mg/g and 42 mg/gfor CBD and Δ⁹-THC respectively), obtained by the official GC-FIDquantitative method. The same semi-quantitative method provided anamount of about 243 and 29 μg/g for CBDP and Δ⁹-THCP respectively.

3.2 Discussion

Up to now, almost 150 phytocannabinoids have been detected in Cannabisplant^(7,33,34), though most of them have neither been isolated norcharacterized. The well-known CBD and Δ⁹-THC have been extensivelycharacterized and proved to possess interesting pharmacologicalprofiles³⁵⁻³⁹, thus the attention towards the biological activity oftheir known homologues like CBDV and Δ⁹-THCV has recently grown asevidenced by the increasing number of publications per year appearing onScopus. Other homologues like those belonging to the orcinoid series arescarcely investigated likely due to their low amount in the plant thatmakes their isolation challenging. In recent years, the agriculturalgenetics research has made great progresses on the selection of rarestrains that produce high amounts of CBDV, CBG and Δ⁹-THCV⁴⁰⁻⁴², thus itwould not be surprising to see in the near future Cannabis varietiesrich in other minor phytocannabinoids. This genetic selection wouldenable the production of extracts rich in a specific phytocannabinoidwith a characteristic pharmacological profile. For this reason, it isimportant to carry out a comprehensive chemical profiling of a medicinalCannabis variety and a thorough investigation of the pharmacologicalactivity of minor and less known phytocannabinoids.

As the pharmacological activity of Δ⁹-THC is particularly ascribed toits affinity for CB₁ receptor, the literature suggests that the lattercan be increased by elongating the alkyl side chain, which representsthe main cannabinoid pharmacophoric driving force¹⁴. Therefore, takingTHC as the lead compound, a series of cannabinoids have been chemicallysynthesized and their biological potency resulted several times higherthan Δ⁹-THC itself¹⁵. To the best of our knowledge, naturally occurringcannabinoids with a linear alkyl side chain longer than five terms havenever been detected or even putatively identified in Cannabis plant.However, the cutting-edge technological platform of the Orbitrap massspectrometry and the use of advanced analytical techniques likemetabolomics can enable the discovery and identification of newcompounds with a high degree of confidence even when present in tracesin complex matrices³4,⁴³. In the present work, we report for the firsttime the isolation and full characterization of two new CBD and Δ⁹-THCheptyl homologs, which we named cannabidiphorol (CBDP) andΔ⁹-tetrahydrocannabiphorol (Δ⁹-THCP), respectively. These common nameswere derived from the traditional naming of phytocannabinoids based onthe resorcinyl residue, in this case corresponding to sphaerophorol.

The biological results obtained in the in vitro binding assay indicatedan affinity for CB₁ receptor more than thirty-fold higher compared tothe one reported for Δ⁹-THC in the literature¹⁴. Also, this encouragingdata was supported by in vivo evaluation of the cannabimimetic activityby the tetrad test, where Δ⁹-THCP decreased locomotor activity andrectal temperature, induced catalepsy and produced analgesia miming theproperties of full CB₁ receptor agonists (FIG. 20). In particular,Δ⁹-THCP proved to be as active as Δ⁹-THC but at lower doses. In fact,the minimum THC dose used in this kind of test is 10 mg/kg, whereasΔ⁹-THCP resulted active at 5 mg/kg in three of the four tetrad tests.These results, accompanied by the docking data, are in line with theextensive structure-activity relationship (SAR) studies performedthrough the years on synthetic cannabinoids, revealing the importance ofthe length of the alkyl chain in position 3 on the resorcinyl moiety inmodulating the ligand affinity at CB₁ receptor.

Although the amount of the heptyl homologues of CBD and Δ⁹-THC in theFM2 variety could appear trifling, both in vitro and in vivo preliminarystudies reported herein on Δ⁹-THCP showed a cannabimimetic activityseveral times higher than its pentyl homologue Δ⁹-THC. Moreover, it isreasonable to suppose that other Cannabis varieties may contain evenhigher percentages of Δ⁹-THCP. It is also important to point out thatthere exists an astonishing variability of subject response to aCannabis-based therapy even with an equal Δ⁹-THC dose⁴⁴⁻⁴⁶. It istherefore possible that the psychotropic effects are due to otherextremely active phytocannabinoids such as Δ⁹-THCP. However, up to nownobody has ever searched for this potent phytocannabinoid in medicinalCannabis varieties. In our opinion, this compound should be included inthe list of the main phytocannabinoids to be determined for a correctevaluation of the pharmacological effect of the Cannabis extractsadministered to patients.

Ongoing studies are devoted to the investigation of the pharmacologicalactivity of CBDP and to expand that of Δ⁹-THCP. It is known that CBDbinds with poor affinity to both CB1 and CB₂ receptors⁴⁷, thus theevaluation of the cannabimimetic activity of CBDP does not appear to beappropriate. On the other hand, more suitable tests should regard itsanti-inflammatory, anti-oxidant and anti-epileptic activity typical ofCBD³⁸. Anyway, the discovery of an extremely potent THC-likephytocannabinoid may shed light on several pharmacological effects notascribable solely to Δ⁹-THC.

3.3 Methods

Plant Material. FM2 Cannabis variety is obtained from the strain CIN-ROproduced by the Council for Agricultural Research and Economics (CREA)in Rovigo (Italy) and provided to the Military Chemical PharmaceuticalInstitute (MCPI, Firenze, Italy) for breeding. FM2 inflorescence (batchn. 6A32/1) was supplied by the MCPI with the authorization of theItalian Ministry of Health (prot. n. SP/062). The raw plant material (10g) was finely grinded and divided into two batches: one batch (500 mg)was extracted with 50 mL of ethanol 96% according to the procedureindicated by the monograph of Cannabis Flos of the GermanPharmacopoeia⁴⁸ and was analyzed by UHPLC-HESI-Orbitrap after properdilution with acetonitrile (×100). The remaining 9.5 g were extractedfollowing the protocol of Pellati et al. with some modifications¹⁸.Briefly, freeze-dried plant material was extracted with 400 mL ofn-hexane for 15 min under sonication in an ice bath. Samples werecentrifuged for 10 min at 2000×g and the pellets were discarded. Theprocedure was repeated twice more. The supernatants were then driedunder reduce pressure and resuspended in 10 mL of acetonitrile, filteredand used for the isolation of CBDPA and THCPA by semi-preparative liquidchromatography.

Isolation of Natural CBDP and Δ⁹-THCP. Aliquots (1 mL) of the solutionobtained as described in the ‘Plant Material’ section were injected in asemi-preparative LC system (Octave 10 Semba Bioscience, Madison, USA).The chromatographic conditions used are reported in the paper by Cittiet al.¹¹. The column employed was a Luna C₁₈ with a fully porous silicastationary phase (Luna 5 μm C18(2) 100 Å, 250×10 mm) (Phenomenex,Bologna, Italy) and a mixture of acetronitrile:0.1% aqueous formic acid70:30 (v/v) was used as mobile phase at a flow rate of 5 mL/min. CBDPAand THCPA (retention time 19.0 min and 75.5 min respectively) wereisolated as reported in our previous work¹¹. The fractions containingCBDBA and THCBA were analyzed by UHPLC-HESI-Orbitrap. The fractionscontaining predominantly either one or the other cannabinoid wereseparately combined and dried on the rotavapor at 70° C. Each residuewas subjected to decarboxylation at 120° C. for two hours in oven. Anamount of about 0.6 mg of CBDP and about 0.3 mg of Δ⁹-THCP wereobtained.

UHPLC-HESI-Orbitrap Metabolomic Analysis. FM2 extracts were analyzed ona Thermo Fisher Scientific Ultimate 3000 system equipped with a vacuumdegasser, a binary pump, a thermostated autosampler, a thermostatedcolumn compartment and interfaced to a heated electrospray ionizationsource and a Q-Exactive Orbitrap mass spectrometer(UHPLC-HESI-Orbitrap). The parameters of the HESI source were setaccording to Citti et al.¹¹: capillary temperature, 320° C.; vaporizertemperature, 280° C.; electrospray voltage, 4.2 kV (positive mode) and3.8 kV (negative mode); sheath gas, 55 arbitrary units; auxiliary gas,30 arbitrary units; S lens RF level, 45. Analyses were acquired usingthe Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, Calif.,USA) in full scan data-dependent acquisition (FS-dd-MS²) in positive(ESI+) and negative (ESI−) mode at a resolving power of 70,000 FWHM atm/z 200. A scan range of m/z 250-400, an AGC of 3e6, an injection timeof 100 ms and an isolation window for the filtration of the precursorions of m/z 0.7 were chosen as the optimal parameters for the massanalyzer. A normalized collision energy (NCE) of 20 was used to fragmentthe precursor ions. Extracted ion chromatograms (EIC) of the [M+H]⁺ and[M−H]⁻ molecular ions were derived from the total ion chromatogram (TIC)of the FM2 extracts and matched with pure analytical standards foraccuracy of the exact mass (5 ppm), retention time and MS/MS spectrum.

The chromatographic separation was carried out on a Poroshell 120 SB-C18(3.0×100 mm, 2.7 μm, Agilent, Milan, Italy) following the conditionsemployed for our previous work¹¹. A semi-quantitative analysis of Δ⁹-THCand CBD and their heptyl analogs CBDP and Δ⁹-THCP was achieved using acalibration curve with an external standard. A stock solution of CBD andΔ⁹-THC, CBDP and Δ⁹-THCP (1 mg/mL) were properly diluted to obtain fivenon-zero calibration points at the final concentrations of 50, 100, 250,500 and 1000 ng/mL for CBD and Δ⁹-THC and of 1, 5, 10, 25 and 50 ng/mLfor CBDP and Δ⁹-THCP. A deuterated standard of Δ⁹-THC-d₃ was added ateach calibration standard at a final concentration of 50 ng/mL. Thelinearity was assessed by the coefficient of determination (R²), whichwas greater than 0.993 for each analyte.

3.4 Synthetic Procedure

All commercially available reagents and solvents were used as purchased,without further purification unless otherwise specified. The followingsolvents have been abbreviated: diethyl ether (Et₂O); dichloromethane(DCM); cyclohexane (CE). Reactions were monitored by thin-layerchromatography on silica gel plates (60F-254, E. Merck) and visualizedwith UV light, or alkaline KMnO₄ aqueous solution. Reaction productswere purified, when necessary, by flash chromatography on silica gel(40-63 m) with the solvent system indicated. NMR spectra were recordedon a Bruker 400 or Bruker 600 spectrometer working respectively at400.134 MHz and 600.130 MHz for ¹H and at 100.62 MHz or 150.902 MHz for¹³C. Chemical shifts (6) are in parts per million (ppm) and they werereferenced to the solvent residual peaks (CDCl₃ δ=7.26 ppm for protonand 6=77.20 ppm for carbon). Coupling constants are reported in hertz(Hz). Splitting patterns are designed following abbreviations are used:singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd),quintet (quin), multiplet (m), broad signal (br s). Monodimensionalspectra were acquired with a spectral width of 8278 Hz (for ¹H-NMR) and23.9 kHz (for ¹³C-NMR), a relaxation delay of 1 s, and 32 and 1024number of transients for ¹H-NMR and ¹³C-NMR, respectively. The COSY wererecorded as a 2048×256 matrix with 2 transients per t1 increment andprocessed as a 2048×1024 matrix. The HSQC spectra were collected as a2048×256 matrix with 4 transients per t1 increment and processed as a2048×1024 matrix, and the one-bond heteronuclear coupling value was setto 145 Hz. The HMBC spectra were collected as a 4096×256 matrix with 16transients per t1 increment and processed as a 4096×1024 matrix, and thelong-range coupling value was set to 8 Hz. Circular dichroism (CD) andUV spectra were acquired on a Jasco (Tokyo, Japan) J-1100spectropolarimeter using a 50 nm/min scanning speed. Quartz cells with a10 mm path length were employed to record spectra in the 500-220 nmrange. Optical rotation (k) was measured with a Polarimeter 240C(cell-length 100 mm, volume 1 mL) from Perkin-Elmer (Milan, Italy).

3.4.1 Synthesis of(1′R,2′R)-4-heptyl-5′-methyl-2′-(prop-1-en-2-yl)-1′,2′,3′,4′-tetrahydro-[1,1′-biphenyl]-2,6-diol,(−)-trans-CBDP

(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol (146 mg, 0.96 mmol,0.9 eq.), solubilized in 15 mL of anhydrous DCM, was added over a periodof 20 minutes to a stirred solution of 5-heptylbenzene-1,3-diol (1) (222mg, 1.07 mmol, 1 eq.) and p-toluenesulfonic acid (20 mg, 0.11 mmol, 0.1eq.) in anhydrous DCM (15 mL) at room temperature and over a positivepressure of argon. After stirring in the same conditions for 1 h, thereaction was quenched with 10 mL of a saturated aqueous solution ofNaHCO₃. The mixture was partitioned between diethyl ether and water. Theorganic layer was separated and washed with brine, dried with anhydrousNa₂SO₄ and evaporated. The residue was chromatographed (ratiocrude:silica 1/120, eluent: CE:DCM 8/2). All the chromatographicfractions were analyzed by HPLC-UV and UHPLC-HESI-Orbitrap and only thefractions containing exclusively CBDP were concentrated to give 76 mg ofa colorless oil (23% yield, purity >99%). ¹H NMR (400 MHz, CDCl₃) δ6.10-6.30 (m, 2H), 5.97 (bs, 1H), 5.57 (s, 1H), 4.66 (s, 1H), 4.66 (bs,1H), 4.56 (s, 1H), 3.89-3.81 (m, 1H), 2.52-2.35 (m, 3H), 2.24 (td,J=6.1, 12.7 Hz, 1H), 2.09 (ddt, J=2.4, 5.1, 17.9 Hz, 1H), 1.89-1.74 (m,5H), 1.65 (s, 3H), 1.55 (qnt, J=7.6 Hz, 2H), 1.28 (td, J=4.7, 8.2, 9.0Hz, 8H), 0.87 (t, J=6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.27,154.09, 149.56, 143.23, 140.22, 124.30, 113.93, 111.01, 109.91, 108.26,46.33, 37.46, 35.70, 31.99, 31.14, 30.59, 29.43, 29.35, 28.60, 23.86,22.84, 20.71, 14.29. HRMS m/z [M+H]⁺ calcd. for C₂₃H₃₅O₂ ⁺: 343.2632.Found: 343.2629; [M−H]⁻ calcd. for C₂₃H₃₃O₂ ⁻: 341.2475. Found:341.2482. [α]_(D) ²⁰=−146° (c=1.0, ACN).

3.4.2 Synthesis of(6aR,10aR)-3-heptyl-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol,(−)-trans-Δ⁸-THCP

The set-up of the reaction for the synthesis of (−)-trans-4⁸-THCP wasperformed as described for (−)-trans-CBDP and the resulting mixture wasstirred at room temperature for 48 hours. The mixture was diluted withdiethyl ether, and washed with a saturated solution of NaHCO₃ (10 mL).The organic layer was collected, washed with brine, dried (anhydrousNa₂SO₄) and concentrated. After purification over silica gel (ratiocrude:silica 1/150, eluent: CE:Et₂O 95.5) 315 mg of a colorless oil (46%yield) were obtained. ¹H NMR (400 MHz, CDCl₃) δ 6.28 (d, J=1.6 Hz, 1H),6.10 (d, J=1.6 Hz, 1H), 5.46-5.39 (m, 1H), 4.78 (s, 1H), 3.20 (dd,J=4.5, 16.0 Hz, 1H), 2.70 (td, J=4.7, 10.8 Hz, 1H), 2.44 (td, J=2.3, 7.4Hz, 2H), 2.21-2.10 (m, 1H), 1.92-1.76 (m, 3H), 1.70 (s, 3H), 1.63-1.52(m, 2H), 1.38 (s, 3H), 1.30 (tt, J=4.3, 9.4, 11.8 Hz, 8H), 1.11 (s, 3H),0.88 (t, J=7.0 Hz, 3H).

3.4.3 Synthesis of(6aR,10aR)-3-heptyl-9-chloro-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol(HCl-THCP)

ZnCl₂ 1N in Et₂O (440 μL, 0.44 mmol, 0.5 eq.) was added to a stirredsolution of Δ⁸-THCP (300 mg, 0.87 mmol, 1 eq.) in 20 mL of anhydrousDCM, at room temperature and under nitrogen atmosphere. After 30minutes, the reaction was cooled at 0° C. and 2 mL of HCl 4N in dioxanewas added. The resulting mixture was stirred at room temperature,overnight and then diluted with diethyl ether. The organic layer wascollected and washed, in sequence, with an aqueous saturated solution ofNaHCO₃ and brine. After dehydration anhydrous Na₂SO₄, the organic phasewas concentrated to give 305 mg (93% yield) of a yellowish oil, pureenough to be used in the next step without further purification. ¹H NMR(400 MHz, CDCl₃) δ 6.24 (d, J=1.7 Hz, 1H), 6.07 (d, J=1.6 Hz, 1H), 4.94(s, 1H), 3.45 (dd, J=2.9, 14.4 Hz, 1H), 3.05 (td, J=2.9, 11.3 Hz, 1H),2.42 (td, J=1.5, 7.4 Hz, 2H), 2.20-2.12 (m, 1H), 1.80-1.71 (m, 1H), 1.66(s, 4H), 1.60-1.51 (m, 2H), 1.49-1.42 (m, 1H), 1.38 (s, 3H), 1.34-1.18(m, 10H), 1.13 (s, 3H), 0.87 (t, J=6.6 Hz, 3H). ESI-MS m/z [M+H]+ calcd.for C₂₃H₃₆ ³⁵[Cl]O₂ ⁺: 379.2. Found: 379.4. Calcd. for C₂₃H₃₆ ³⁷[Cl]O₂⁺: 381.2. Found: 381.3.

3.4.4 Synthesis of(6aR,10aR)-3-heptyl-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol,(−)-trans-Δ⁹-THCP

HCl-THCP (305 mg, 0.82 mmol, 1 eq.) was solubilized in 10 mL ofanhydrous toluene and cooled at −15° C. 1.75 N potassium t-amilate intoluene (1.17 mL, 2.05 mmol, 2.5 eq.) was added dropwise with a syringeto the first solution under a positive pressure of argon. The mixturewas stirred in the same condition for 15 minutes and then at 60° C. for1 h. After cooling at room temperature, the reaction was quenched with a1% solution of ascorbic acid and diluted with diethyl ether. The organiclayer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated. The residue was chromatographed (ratio crude/silica 1:300,hexane:i-propyl ether 9.1) to give 232 mg of a greenish oil (83% yield).50 mg of (−)-trans-Δ⁹-THCP were further purified by semipreparative HPLCto prepare a pure analytic standard (purity >99.9%). ¹H NMR (600 MHz,CDCl₃) δ 6.30 (t, J=2.0 Hz, 1H), 6.27 (d, J=1.6 Hz, 1H), 6.14 (d, J=1.5Hz, 1H), 4.75 (s, 1H), 3.20 (dt, J=2.5, 10.8 Hz, 1H), 2.43 (dd, J=6.4,8.9 Hz, 2H), 2.22-2.11 (m, 2H), 1.97-1.87 (m, 1H), 1.69-1.65 (m, 4H),1.58-1.50 (m, 2H), 1.43-1.37 (m, 4H), 1.34-1.21 (m, 8H), 1.09 (s, 3H),0.87 (t, J=6.6 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 154.97, 154.34,143.02, 134.59, 123.92, 110.30, 109.22, 107.72, 77.38, 46.01, 35.72,33.78, 31.99, 31.37, 31.16, 29.50, 29.38, 27.77, 25.22, 23.55, 22.87,19.47, 14.29. HRMS m/z [M+H]⁺ calcd. for C₂₃H₃₅O₂ ⁺: 343.2632. Found:343.2633; [M−H]⁻ calcd. for C₂₃H₃₃O₂ ⁻: 341.2475. Found: 341.2481.[α]_(D) ²⁰=−166° (c 1.0, ACN).

3.5 Binding at CB1 and CB2 Receptors

The binding affinity of (−)-trans-Δ⁹-THCP against human CB₁ and CB₂receptors was assessed by Eurofins Discovery using a radioligand bindingassay. Ten concentrations of the phytocannabinoid from 1 nM to 30 μMwere tested in duplicate. [3H]CP55940 (at 2 nM, K_(d)=2.4 nM) and[³H]WIN 55212-2 (at 0.8 nM, K_(d)=1.5 nM) were used as specificradioligand for hCB₁ and hCB₂, respectively^(49,50). Eq. 1 was employedto calculate the percent inhibition of control specific binding obtainedin the presence of the tested compounds.

$\begin{matrix}{{\%\mspace{14mu}{in}} = {100 - \left( {\frac{{measured}\mspace{14mu}{specific}\mspace{14mu}{binding}}{{control}\mspace{14mu}{specific}\mspace{14mu}{binding}}*100} \right)}} & {{eq}.\mspace{11mu} 1}\end{matrix}$

A non-linear regression analysis of the competition curves generatedwith mean replicate values (eq. 2) was used to calculate the IC₅₀ values(concentration causing a half-maximal inhibition of control specificbinding)⁵¹.

$\begin{matrix}{Y = {D + \left\lbrack \frac{A - D}{1 + {\left( \frac{C}{C50} \right){nH}}} \right\rbrack}} & {{eq}.\mspace{11mu} 2}\end{matrix}$

where Y is the specific binding, A is the left asymptote of the curve, Dis the right asymptote f the curve, C is the compound concentration, C₅₀is the IC₅₀ value and nH is the slope factor. This analysis was carriedout using a software developed at Cerep (Hill software) and validated bycomparing the data with that generated by the commercial softwareSigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.). The inhibitionconstants (K_(i)) were determined using the Cheng Prusoff equation (eq.3):

$\begin{matrix}{{Ki} = \frac{I\; C_{50}}{\left( {1 + \frac{L}{K_{D}}} \right)}} & {{eq}.\mspace{11mu} 3}\end{matrix}$

where L is the concentration of the radioligand, and K_(D) is theaffinity of the radioligand for the receptor.

The data obtained for CP 55940 (CB₁ IC₅₀=1.7 nM, CB₁ K_(i)=0.93 nM) andWIN 55212-2 (CB₂ IC₅₀=2.7 nM, CB₂ K_(i)=1.7 nM) were in accordance withthe values reported in literature^(49,50).

Docking simulation. The prediction of the binding mode of THCP incomplex with human CB₁ receptor was performed using Maestro 10.3 of theSchrödinger Suite⁵². The crystallographic structure of the activeconformation of CB₁ in complex with AM11542 (PDB ID: 5XRA) wasdownloaded from the Protein Data Bank and was used as reference fordocking calculation. The protein was prepared using the ProteinPreparation Wizard module⁵³. The protonation and tautomeric states ofthe residues were adjusted at pH 7.0, water molecules were removed, andthe hydrogens position was minimized with the OPLS_2005 force field. Thechemical structure of (−)-trans-Δ⁹-THCP were sketched with ChemDraw 12.0and converted from 2D to 3D with the LigPrep utility⁵⁴. Fiveconformations per ligand were initially generated, and appropriateionization state and tautomers were evaluated for each conformation atphysiological pH^(55,56). Afterwards, ligand conformations wereminimized with the OPLS_2005 force field. Rigid docking was performed inextra precision mode with Glide version 6.857.

3.6 Tetrad Test

Male C57BL6/J mice (7 weeks old; n=5) were treated with Δ⁹-THCP (10, 5and 2.5 mg/kg) or vehicle (1:1:18; ethanol:Kolliphor EL:0.9% saline) byi.p. administration. Mice were evaluated for hypomotility (open fieldtest), hypothermia (body temperature), antinociceptive (hot plate test),and cataleptic (bar test) effects, using the tetrad tests⁵⁸. Statisticalanalysis was performed using the Kruskall-Wallis test and Dunn's posthoc tests.

Body temperature. The mouse was immobilized and the probe gentlyinserted for 1 cm into the rectum until stabilization of temperature.Between each mouse the probe was cleaned with 70% ethanol and dried withpaper towel.

Open field. The open field test was used for the evaluation of motoractivity. Behavioral assays were performed 30 min after drug (orvehicle) injection. The apparatus was cleaned before each behavioralsession by solution of 70% ethanol. Naïve mice were randomly assigned toa treatment group. Behaviors were recorded, stored, and analyzed usingan automated behavioral tracking system (Smart v3.0, Panlab HarvardApparatus). Mice were placed in an OFT arena (l×w×h: 25 cm×25 cm), andambulatory activity (total distance travelled in centimeter), wererecorded for 15 minutes and analyzed.

Bar test. The bar test was used for the evaluation of catalepsy. Bar wasa 40 cm in length and 0.4 cm in diameter glass rod, which washorizontally elevated by 5 cm above the surface. Both forelimbs of micewere positioned on the bar and its hind legs on the floor of the cage,ensuring that the mouse was not lying down on the floor. The chronometerwas stopped when the mouse descends from the bar (i.e., when the twoforepaws touched the floor) or when 10 min have elapsed (i.e., cut-offtime). Catalepsy was measured as the time duration each mouse held theelevated bar by both his forelimbs (latency for moving in seconds).

Hot plate. Changes in nociception were evaluated by the hot plate test.On the day of experiment each mouse was placed on a hot plate (UgoBasiele) at a constant temperature of 52° C. Licking of the hind paws,as well as jumping, were considered as a nociceptive response (NR) andthe latency was measured in seconds 85 minutes after drug or vehicleadministration. The latency to the NR was recorded and a 30 or 60 scut-off time was used in order to prevent tissue damage.

3.7 References

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While the present disclosure has been discussed in terms of certainembodiments, it should be appreciated that the present disclosure is notso limited. The embodiments are explained herein by way of example, andthere are numerous modifications, variations and other embodiments thatmay be employed that would still be within the scope of the presentdisclosure.

What is claimed is:
 1. A cannabinoid compound selected from the groupconsisting of: (−)trans (1R,6R) cannabidibutol [CBDB] of formula (I),(−)trans (1R,6R) Δ⁹ tetrahydrocannabutol [Δ⁹ THCB] of formula (II),(−)trans (1R,6R) Δ⁹ tetrahydrocannabiphorol [Δ⁹ THCP] of formula (III),(−)trans (1R,6R) cannabidiphorol [CBDP] of formula (IV)

wherein R₁ is —H, or their corresponding acid derivatives wherein R₁ is—COOH.
 2. A method to produce the cannabinoid compound CBDB of formula(I) of claim 1, comprising reacting(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol with5-butylbenzene-1,3-diol according to scheme (I) in presence of an acidiccatalyst, obtaining CBDB.


3. A method to produce the cannabinoid compound CBDP of formula (IV) ofclaim 1, comprising reacting(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol with5-heptylbenzene-1,3-diol according to scheme (II) in presence of anacidic catalyst, obtaining CBDP.


4. The method of claim 2, wherein the acidic catalyst isp-toluensulphonic acid.
 5. The method of claim 3, wherein the acidiccatalyst is p-toluensulphonic acid.
 6. The method of claim 2, whereinthe reaction is performed under inert atmosphere in a halogenatedorganic solvent at a temperature of −10±5° C., for a time ranging from30 to 90 min.
 7. The method of claim 3, wherein the reaction isperformed under inert atmosphere in a halogenated organic solvent at atemperature of −10±5° C., for a time ranging from 30 to 90 min.
 8. Amethod to produce the cannabinoid compound Δ⁹ THCB of formula (II) ofclaim 1, comprising reacting the CBDB of formula (I) of claim 1 withhydrochloric acid obtaining (−)trans HCl-THCB of formula (V),

followed by treating the compound (V) with a basic compound, obtainingΔ⁹ THCB.
 9. A method to produce the cannabinoid compound Δ⁹ THCP offormula (III) of claim 1, comprising reacting the CBDP of formula (IV)of claim 1 with hydrochloric acid obtaining (−)trans HCl-THCP of formula(VI),

followed by treating the compound (VI) with a basic compound, obtainingΔ⁹ THCP.
 10. The method of claim 8, wherein said CBDB is in a mixturewith (−)trans Δ⁸ THCB of formula (VII) (VII)


11. The method of claim 9, wherein said CBDP is in a mixture with(−)trans Δ⁸ THCP of formula (VIII)


12. The method of claim 8, wherein said basic compound is potassiumamylate.
 13. The method of claim 9, wherein said basic compound ispotassium amylate.
 14. A method of treating a disease mediated by theCB1 receptor comprising administering a compound claim 1 to a patient inneed thereof.
 15. A pharmaceutical composition comprising a compound ofclaim 1 in presence of one or more pharmaceutically acceptableexcipients.